Agricultural and Food Science in Finland 365 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 8 (1999): 365–392. © Agricultural and Food Science in Finland Manuscript received January 1999 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 8 (1999): 365–392. Review Association between protein feeding and reproductive efficiency in the dairy cow: specific emphasis on protein feeding in Finland Kevin John Shingfield Agricultural Research Centre of Finland, Animal Production Research, FIN-31600 Jokioinen, Finland, e-mail: kevin.shingfield@mtt.fi Marjatta Jokela Valio Ltd, Farm Services and Member Relations, PO Box 30, FIN-00039 Helsinki, Finland. Current address: Ministry of Agriculture and Forestry, Department of Agriculture, PO Box 232, FIN-00171 Helsinki, Finland Kaisa Kaustell and Pekka Huhtanen Agricultural Research Centre of Finland, Animal Production Research, FIN-31600 Jokioinen, Finland Juha Nousiainen Valio Ltd, Farm Services and Member Relations, PO Box 30, FIN-00039 Helsinki, Finland Associations between protein feeding and reproductive efficiency in the dairy cow are reviewed. Examination of published data indicated that reproductive responses assessed as days open, services per conception or conception rate following changes in protein feeding tend to be inconsistent. Dis- crepancies can arise due to between-study variations in experimental design, statistical analysis, sample population size, uterine health, cow age, parity, reproductive management or nutrient intake. Detri- mental effects on reproductive efficiency following periods of excessive protein feeding are often attributed to increases in tissue urea and ammonia concentrations leading to impaired reproductive physiology, modified endocrine function or exacerbated postpartum negative energy balance. Exam- ination of data collected from Finnish dairy herds (n = 16 051) participating in the national milk recording scheme during 1993 indicated that milk production was maximised in herds fed diets con- taining 180 g crude protein/kg dry matter. In contrast, no consistent relationships were identified between increases in on-farm protein feeding necessary to secure higher milk production and herd reproductive efficiency assessed as calving interval, first service interval and number of insemina- tions per calving. Further examination of data derived from 5 437 herds within the National record- ing scheme indicated that on-farm reproductive efficiency was independent of large variations in the mean annual urea concentration of bulk tank milk. It is concluded that increases in the crude protein content of Finnish dairy cow rations from 150 to between 170 and 180 g/kg dry matter would allow improvements in milk production to be realised without leading to significant reductions in repro- ductive efficiency. Key words: dairy cow, dietary protein, reproductive efficiency, urea 366 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Shingfield, K.J. et al. Protein feeding and reproductive efficiency Introduction Over recent years considerable emphasis has been placed on protein feeding of the dairy cow, since protein is typically the most expensive feed ingredient, milk payments tend to favour the pro- duction of milk protein and excessive nitrogen (N) excretion has a detrimental affect on the en- vironment (Broderick and Clayton 1997). Pro- ducers attempting to increase milk production, may consider the use of protein supplements, since increases in dietary crude protein (CP) content often elicit positive milk production re- sponses (e.g. Thomas and Rae 1988, Chamber- lain et al. 1989). Recently, Huhtanen (1998a) using data from 7 Finnish production studies reported that dietary inclusion of 1 kg (on an air- dry basis) of rapeseed meal elicited mean milk and milk protein yield responses of 1050 and 39.4 g/d, respectively. Protein feeding also has an im- pact on reproductive efficiency, and has been re- ported to be decreased in cows fed diets con- taining either low (e.g. Hibbitt 1984) or high (e.g. Edwards et al. 1980, Kaim et al. 1983, Canfield et al. 1990) protein contents. Supplementary pro- tein feeding equivalent to increases in dietary concentrations of between 30 and 65 g/kg dry matter (DM) have often been associated with depressions in reproductive efficiency (e.g. Jor- dan and Swanson 1979a, Folman et al. 1981, Chalupa 1984, Canfield et al. 1990). However, reproductive efficiency has been also been re- ported to be unaffected by increases in dietary CP content of 50 g/kg DM (Howard et al. 1987) and 70 g/kg DM (Carroll et al. 1988). Following ingestion by ruminant animals, di- etary CP is catabolised to ammonia via microbial proteases. Intakes of rumen degradable or unde- gradable protein in excess of requirements can result in increases in ammonia, urea and other unidentified nitrogenous compound concentra- tions in body tissues. Increased concentrations of nitrogenous compounds liberated during rumen and tissue nitrogen metabolism in the reproduc- tive tract have been considered to exert detrimen- tal affects on reproductive organ function and modify tissue responses to reproductive hormones (Ferguson and Chalupa 1989). Overfeeding of protein has also been suggested to indirectly im- pair reproductive performance by exacerbating negative energy balance during early lactation due to increased energy requirements for ureogenesis (Chalupa 1984, Oldham 1984). The aim of the current paper is to review the relationship between protein feeding and repro- ductive efficiency in the dairy cow and the po- tential underlying physiological mechanisms accounting for altered reproductive responses. Based on data collected from the national milk recording scheme during 1993, the potential impact of increases in on-farm protein feeding and associated changes in the urea concentra- tion of bulk tank milk on the reproductive effi- ciency of Finnish dairy herds was assessed. Protein feeding During the last decade several new protein eval- uation systems have been proposed (e.g. Agri- cultural and Food Research Council 1992, Mad- sen et al. 1995) in order to improve the accuracy of protein feeding to ruminant animals by ac- counting for both microbial and host tissue N metabolism. A common feature of new protein evaluation systems is the differentiation of die- tary N that is degraded in the rumen, and that which escapes degradation and enters the small intestine for subsequent digestion and absorp- tion. Despite variations in the proportion of N in the form of either true or non-protein N be- tween ruminant feeds, protein systems currently evaluate feeds in terms of total N or CP content defined as 6.25 x N. Measurement of rumen nitrogen degradability Once ingested, dietary CP is subjected to micro- bial catabolism the extent of which is dependent 367 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 8 (1999): 365–392. upon rumen environment and the physical prop- erties of a particular protein (Czerkawski 1986). Obtaining reliable estimates of the extent of N degradation by rumen microbes is extremely dif- ficult, due to variations both within and between feeds and the rumen environment (Cottrill 1993). Several methods have been used to provide esti- mates of rumen N degradability. In vivo meas- urements of degradable N calculated as the dif- ference between N intake and the sum of endog- enous and microbial N entering the small intes- tine, are scientifically the most sound but require the use of surgically modified animals and mi- crobial and digesta markers. Although in-vivo procedures are subject to error due to problems associated with the determination of microbial N flow (Broderick and Merchen 1992), and are inappropriate for routine feed degradability measurements they are necessary to provide ref- erence values in order to validate alternative in vitro methods (NRC 1985). The in situ method is probably the most wide- ly used procedure to determine rumen N degrad- ability. This approach proposed by Mehrez and Ørskov (1977) involves incubating feed samples in nylon bags and suspending them for a period of time (typically 48 h) in the rumen of sheep or cattle. Rumen N degradability is estimated as the difference in bag N content following incuba- tion, and allows the rate of N disappearance of an individual feed to be determined. Combining the rate of N disappearance and an appropriate estimate of rumen outflow rate allows the effec- tive rumen N degradability (Ørskov and McDon- ald 1979) to be estimated. With the exception of the protein evaluation system adopted in France, tabulated values of feed N degradability neces- sary to assist formulation of ruminant diet have been estimated using the in situ method. Despite being widely used the method does have limita- tions. Criticisms levelled at the technique include the lack of reduction in incubated feed particle size through chewing and rumination, an incor- rect assumption that N disappearing from the bag at 0 h incubation time is immediately degraded and insufficient method standardisation (Nocek 1988, Michalet-Doreau and Ould-Bah 1992). Huhtanen et al. (1998) recently demonstrated that microbial enzyme activities inside nylon bags can be as low as proportionately 0.10 of that in rumen ingesta. Requirements for surgically modified ani- mals and associated limitations in terms of time and cost mean that both in vivo and in situ meth- ods cannot be considered for routine evaluation of feed N degradability (Cottrill 1993). Conse- quently there has been drive to develop in vitro methods to estimate N degradability of a feed. In vitro approaches can be characterised as ei- ther attempting to determine N solubility in var- ious solvents based on the assumption that this is closely related to rumen degradability or the simulation of N degradation by incubation of feeds with either mixed or single strains of bac- teria. The merits and demerits of the in vitro ap- proach to estimate N degradability are well documented (refer to NRC 1985, Nocek 1988, Cottrill 1993). Protein feeding in Finland Dairy cow diets tend to be formulated from a wide range of ingredients, due to variations in feed cost and availability and the value of milk within milk payment schemes. Consequently, relatively large differences in the protein con- tent of dairy cow rations can exist between indi- vidual countries. Finnish dairy cow rations typ- ically contain lower amounts of protein (150 g CP/kg DM; Kaustell et al. 1998) than diets fed in other European countries, such as Holland (185–220 g/kg DM, Tamminga 1992) or the United Kingdom (180–190 g/kg DM, N.W. Of- fer, personal communication). Formulation of ruminant diets typically in- volves a computer assisted coupling of calculat- ed protein requirements with tabulated feed val- ues. In Finland, protein feeding of ruminant an- imals based on digestible crude protein (ARC 1965) was replaced in 1995 by a new system that defines dietary protein in terms of amino acids absorbed in the small intestine (AAT) and pro- tein balance in the rumen (PBV; Tuori et al. 368 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Shingfield, K.J. et al. Protein feeding and reproductive efficiency 1998). The term AAT describes the amount of amino acids derived from microbial protein and undegraded feed protein digested in the small intestine, while PBV describes rumen N availa- bility relative to microbial requirements. Protein feeding recommendations based on the Finnish AAT-PBV system differ from those calculated according to AAT-PBV systems (Mad- sen et al. 1995) adopted in other Nordic coun- tries. Differences arise because dietary digesti- ble carbohydrate and rumen degradable protein content are used to predict microbial protein sup- ply and lower feed particle outflow rates from the rumen are used to calculate effective rumen protein degradability. Incorporating these mod- ifications has been shown to allow accurate pre- dictions of dietary protein value (Tuori et al. 1998). Protein values assigned to diets accord- ing to the Finnish AAT-PBV system have been shown to be closely correlated (r = 0.977, n = 67) with yields of milk protein (Huhtanen 1998b), indicating that production responses fol- lowing changes in protein feeding can be pre- dicted accurately. There is however, also an ur- gent need to establish potential effects of pro- tein feeding on reproductive efficiency, to en- sure that protein intakes considered optimal for milk production are not associated with signifi- cant depressions in reproductive performance. Association between protein feeding and reproductive efficiency The association between protein feeding of ru- minant livestock and reproductive performance is a concern in many countries. However there is a clear distinction between Third World and developed countries. In most developing coun- tries ruminants have to survive on poor quality feeds which are deficient in numerous nutrients, particularly protein. As a result, reproductive efficiency of ruminant livestock in these coun- tries is generally constrained by a deficiency in dietary protein supply (Kaur and Arora 1995). In developed countries, the situation is reversed such that excessive intakes of dietary protein have often been implicated in situations of re- productive inefficiency (e.g. Canfield et al. 1990, Elrod and Butler 1993). Protein deficiency Since reproduction responses to protein deficien- cy have been considered in detail in several pub- lished reviews (Kaur and Arora 1995, Robinson 1990) associations between reproductive effi- ciency and low dietary protein intakes are dis- cussed in brief. Provision of an adequate supply of nutrients during the pre and postpartum peri- od is paramount to securing reproductive per- formance. It is well established that optimal ovu- lation rates can only be achieved when animals receive sufficient supplies of energy and protein (Henniawati and Fletcher 1986). A deficiency of dietary protein can result in poor reproductive performance due to ovarian dysfunction or in- creases in embryo mortality. Due to disturbances of ovarian function, protein deficiency is often manifested as protracted postpartum anoestrus or as a delayed or extended oestrus. Protein de- ficiency can markedly increase anoestrus in crossbred cows by as much as 140 d (Juneja and Arora 1989). Protracted anoestrus represents a major cause of reproductive inefficiency, such that reducing the interval between parturition and rebreeding has been suggested to be the most feasible means of improving reproductive effi- ciency in cattle (Robinson 1990). Feeding diets containing sufficient protein is a prerequisite for reducing the parturition-rebreeding interval, since oestrus is often delayed in protein deficient animals. Sasser et al. (1988) noted that the inci- dence of behavioural oestrus was reduced by 29% and delayed on average by 11 d in primipa- rous beef cows fed diets deficient in protein (77 g CP/kg DM) compared to control diets contain- ing 225 g CP/kg DM. In addition to delayed re- sumption of normal oestrus cyclicity, protein deficiency reduced conception rate at first serv- 369 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 8 (1999): 365–392. ice (25 and 71% for low and high protein diets, respectively) and the incidence of pregnancy (re- spective values of 32 and 74%). Cows deficient in protein can also develop hypoalbuminaemia a condition inversely relat- ed to the number of services per conception (Payne et al. 1970). Protein feeding is also cen- tral to embryo survival and foetal development, since inadequate supplies have been reported to cause a significant reduction in embryo survival and development (Folman et al. 1983, Robinson 1986, Kaur and Arora 1995). Excessive protein intake In order to attain high levels of milk production, dairy cows in developed countries are typically fed high CP diets. Use of high protein intakes to promote milk production can however, have ad- verse effects on subsequent reproduction (Old- ham 1984). It is becoming increasing apparent that high as well as low intakes of dietary pro- tein appear to be associated with reproductive disturbances. In developed countries, impaired reproductive performance is more often associ- ated with excessive intakes of protein (Kaur and Arora 1995). Feeding excessive amounts of protein to dairy cows can impair reproductive efficiency with- out inducing changes in oestrus (Kaim et al. 1983). Canfield et al. (1990) reported that con- ception rate at first service was significantly re- duced in cows fed high protein diets (192 g CP/ kg DM) compared to animals fed diets contain- ing only moderate levels (165 g CP/kg DM). Impaired reproduction efficiency following changes in dietary CP content may be associated with changes in uterine environment, since die- tary CP content had only minor effects on days to first ovulation, days to first service or lutein- izing hormone secretion. However, diets defi- cient in energy can also lead to decreased con- ception rates (e.g. Butler and Elrod 1991). Examination of published data indicates that feeding diets containing large amounts of CP is often associated with increases in days open and services per conception (Table 1). However, the extent of these responses to increases in dietary CP content tend to be inconsistent, and in some cases increases in dietary CP content have re- duced the number of services per conception (Chandler et al. 1976, Hibbitt 1984). Increases in dietary protein content have also been nega- tively associated with conception rate, but as with other measures of reproductive efficiency these responses tend to be inconsistent (Fig. 1). Most studies of reproductive efficiency in the dairy cow have identified a negative association with increases in protein feeding (e.g. Kaim et al. 1983, Canfield et al. 1990). In contrast, other studies have reported that feeding diets contain- ing CP concentrations excess of 195 g/kg DM has only minor effects on conception rate (Howard et al. 1987, Carroll et al. 1988, Bruck- ental et al. 1989, Barton et al. 1996). Protein degradability Detrimental effects of excessive protein feeding on reproductive efficiency have often been linked with the intake of CP that is degraded in the rumen (Folman et al. 1981, Ferguson et al. 1988, Canfield et al. 1990, Butler 1998). Based on one study, Carroll et al. (1988) proposed that reproductive management, rather than the intake of dietary CP either degraded or escaping rumen catabolism was more likely to account for vari- ations in reproductive efficiency. However, this suggestion is inconsistent with data of Elrod and Butler (1993) indicating that excessive intakes of rumen degradable protein decreased reproduc- tive efficiency of cows managed under condi- tions considered optimal. Reproductive efficiency, expressed as the proportion of primiparous beef cattle served dur- ing first oestrus has also been shown to improve following postpartum protein feeding in excess of predicted requirements, provided it escapes extensive degradation in the rumen (Wiley et al. 1991). In another study with beef cattle, Rusche et al. (1993) reported that reproductive perform- ance was independent of changes in protein 370 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Shingfield, K.J. et al. Protein feeding and reproductive efficiency Table 1. Associations between dietary crude protein content with days open and services per conception reported in the literature. Reproduction Dietary crude protein content (g/kg DM) Reference parameter 120–145 145–165 165–190 190–220 Days open 130 140 Chandler et al. 1976 69 96 106 Jordan and Swanson 1979a 123 141 139 Edwards et al. 1980 84–98 102 Folman et al. 1981 82 127 Piatkowski et al. 1981 80 80 Howard et al. 1987 72 82 Carroll et al. 1988 71 81 Barton et al. 1996 Services per conception 2.4 2.1 Chandler et al. 1976 1.5 1.9 2.5 Jordan and Swanson 1979a 2.3 2.6 2.7 Edwards et al. 1980 1.5–1.8 2.3 Folman et al. 1981 2.0 2.8 Piatkowski et al. 1981 1.8 2.3 Kaim et al. 1983 1.4 1.4 Howard et al. 1987 1.5 1.8 Carroll et al. 1988 2.3 2.1–2.7 Bruckental et al. 1989 1.2 1.6 Elrod and Butler 1993 1.7 1.8 Barton et al. 1996 Fig. 1. Association between die- tary crude protein content and con- ception rate reported in the litera- ture. Data derived from Jordan and Swanson 1979a (+), Folman et al. 1981 (�), Piatkowski et al. 1981 (o), Kaim et al. 1983 (+), Howard et al. 1987 (▲), Carroll et al. 1988 (●), Bruckental et al. 1989 (�), Canfield et al. 1989 (�), Elrod and Butler 1993 (�) and Barton et al. 1996 (�). source or intake. Based on their findings, Elrod and Butler (1993) proposed that depressions in reproductive efficiency associated with exces- sive intakes of degradable protein were mediat- ed via an unidentified mechanism leading to a decrease in uterine pH during the luteal phase. 371 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 8 (1999): 365–392. Further studies have shown that high intakes of rumen degradable protein before ovulation and during early pregnancy period can adversely af- fect embryo survival in non-lactating ewes (Bis- honga et al. 1994), but not in all cases. Wallace et al. (1996) reported that feeding additional ru- men degradable nitrogen in the form of urea sup- plements during pre, peri and post-ovulation periods had no influence on fertilisation rate, embryo survival or luteotropic protein secretion. In contrast, a more recent study demonstrated that feeding excess rumen degradable protein decreased ovarian follicular development, de- layed postpartum luteal activity and reduced the accumulation of luteal tissue (Garcia-Bojalil et al. 1998). However, these findings are equivo- cal because dietary supplements of rumen pro- tected long chain fatty acids leading to an in- crease in energy intake augmented the decline in luteal tissue development, doubled the number of corpora lutea and reduced the interval to the first rise in progesterone by 6d. Furthermore, the inclusion of protected fat was also associated with an increase in the incidence of pregnancy from 52 to 86%, suggesting that negative effects attributed to rumen degradable protein may have reflected changes in the extent of energy defi- ciency or energy status. Feeding management Carroll et al. (1988) proposed that the method of feeding may indirectly influence reproductive efficiency, since rumen ammonia concentrations may be more stable in cows fed a total mixed ration, preventing surges of ammonia entering the peripheral circulation. Hepatic extraction and metabolic conversion of ammonia absorbed from the rumen appears to be more efficient when ru- men ammonia concentrations accumulate stead- ily over time compared to rapid increases. Con- tinuous intraruminal infusions of urea in dairy cows have been shown to elicit progressive in- creases in urea concentrations of peripheral blood, but concentrations of ammonia have re- mained unaffected until the capacity for hepatic conversion is exceeded (Choung et al. 1990). In the same study, twice-daily administration of non-toxic urea doses caused ammonia concen- trations in peripheral blood to increase substan- tially 3–4 h post infusion. Increases in the ap- pearance of ammonia in peripheral blood are thought to occur as a result of ‘leakage’ through the liver (Bartley et al. 1981) or transfer of am- monia via non-hepatic routes (Chalmers et al. 1976). Protein contained in grass silage is rapidly and extensively degraded in the rumen (Huh- tanen 1998a) and digestion of diets containing large proportions of grass silage is characterised by an inefficient rumen microbial utilisation of silage N, leading to substantial amounts of am- monia absorption from the rumen (Chamberlain et al. 1986). Consequently, a clear association between reproductive efficiency and silage qual- ity might be expected. Gustafsson and Carlsson (1993) conducted a field study with 29 Swedish dairy herds and reported that feeding silage with high ammonia concentrations increased the in- terval between calving and conception, but not the interval between calving and first service. Based on these findings they concluded that pro- tein quality could be at least as important as di- etary CP content in determining reproductive efficiency. However, reduced reproductive re- sponses attributed to ammonia content and hence silage protein quality may be better explained by associated reductions in silage DM intake leading to an exacerbation of postpartum nega- tive energy balance or as a result of potentially toxic amine intakes (Tveit et al. 1992). Due to the general assumption that microbi- al synthesis is often constrained by a mismatch in rumen energy and N availability for microbi- al growth there has been considerable interest in the potential of manipulation of the rate of carbohydrate fermentation to improve the effi- ciency of N capture by rumen microbes. Theo- retically at least, manipulating the rate of rumen carbohydrate fermentation by inclusion of dif- ferent ingredients in ruminant rations is extreme- ly attractive since it may allow the potential neg- ative impact of high intakes of rumen degrada- 372 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Shingfield, K.J. et al. Protein feeding and reproductive efficiency ble protein on reproductive efficiency to be min- imised. However, experimental evidence used to support an increase in microbial production at- tributable to improved synchrony of energy and N release in the rumen (e.g. McCarthy et al. 1989, Sinclair et al. 1993) is questionable. The major criticism of these studies is that interpre- tation of differences in the degree of rumen en- ergy and N synchronicity is confounded with the use of different ingredients to formulate experi- mental diets (Chamberlain and Choung 1995). Subsequent experiments have lead to the gener- al conclusion that there is no real advantage in attempting to enhance microbial protein supply by synchronising energy and N release in the rumen (Henning et al. 1993, Kim et al. 1999). Henning et al. (1993) suggested that formula- tion of ruminant diets should be directed towards providing an even release of energy in the ru- men. In the absence of direct experimental evi- dence to the contrary, the scope of feeding strat- egies attempting to optimise or maintain repro- ductive efficiency by manipulation of the rate of energy and N release in the rumen appears to be limited. Negative energy balance Dietary deficiencies of energy and/or protein are the most common predisposing factors for pro- longing the interval between calving and first oestrus (Kaur and Arora 1995). Nutrient supply has a major role in the initiation of postpartum ovarian cyclicity. During early lactation, energy intake is insufficient to meet energy requirements for maintenance and potential milk production (Garnsworthy 1988). Deficits in energy intake are to some extent compensated for by tissue catabolism, principally intermuscular, subcuta- neous and internal fat stores (Butler-Hogg et al. 1985). When energy expenditure exceeds ener- gy intake a situation generally referred to as neg- ative energy balance exists, and typically reach- es a maximum during the first 7 to 14 d postpar- tum and subsequently recovers at a variable rate. Periods of negative energy balance also tend to coincide with the time of first insemination nec- essary to secure pregnancy during early lacta- tion, and appear to be directly related to first service intervals and lowered conception rates (Butler and Smith 1989). Initiation of the ovari- an cycle tends to occur once the energy nadir returns towards balance (Butler et al. 1981), such that the extent and duration of negative energy balance is currently thought to regulate the re- turn of normal ovarian activity following calv- ing. High yielding dairy cows can remain in neg- ative energy balance during the first 70 d of lac- tation (Villa-Godoy et al. 1988). Onset of post- partum ovulation has been suggested to be de- layed by 0.67 d per MJ of negative energy status experienced during the first 20 d postpartum (Butler and Smith 1989). Energy deficiency (Vil- la-Godoy et al. 1988, Butler and Smith 1989) or loss of live weight (McClure 1970, Heinonen et al. 1988) at service can impair reproductive ef- ficiency. Chalupa (1984) and Oldham (1984) consid- ered that excess protein feeding exerts deleteri- ous effects on fertility by exacerbating negative energy balance during early lactation, since he- patic conversion of ammonia to urea would in- crease metabolisable energy requirements by 48 KJ/g excess N (NRC 1988). Despite limited ex- perimental evidence the role of excessive pro- tein intake on negative energy balance has gen- erally been accepted and often suggested to ac- count for differential reproductive responses to increases in protein feeding (e.g. Ferguson and Chalupa 1989, Kaur and Arora 1995, Butler 1998). However, a number of experimental ob- servations tend to challenge this hypothesis, since there is overwhelming evidence that in- creases in dietary CP content generally improve DM intake and often diet digestibility (e.g. Old- ham 1984, Thomas and Rae 1988, Chamberlain et al. 1989, Huhtanen 1998a). Furthermore, pro- tein supplementation of grass silage based diets has also been observed to improve live weight gain (Huhtanen and Heikkilä 1996) but have no effect on plasma β-hydroxybutyrate (Heikkilä et al. 1998) or non-esterified fatty acid concentra- tions (Rinne et al. 1995), findings inconsistent 373 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 8 (1999): 365–392. with exacerbated postpartum negative energy balance. Secretion of insulin, a major metabolic reg- ulator of plasma glucose concentration is highly dependent on nutrient supply, being modified in the presence of various metabolic stimuli includ- ing glucose, volatile fatty acids and amino acids (Brockman and Laarveld 1986). Glucose is a more potent stimulator of insulin secretion than propionate the major gluconeogenic substrate in ruminants (Kelly et al. 1993) while increases in ammonia in peripheral blood appear to be asso- ciated with reduced insulin and increased glu- cose concentrations (Choung et al. 1990). Dur- ing periods of postpartum negative energy bal- ance, plasma insulin and glucose concentrations tend to become depressed (Ferguson and Cha- lupa 1989), and can potentially modify hypoth- alamo-hypophyseal-ovarian axis function (e.g. Rutter and Manns 1987). Tissue culture studies have demonstrated that increases in insulin con- centration can stimulate follicle stimulating hor- mone secretion by pituitary cells (Adashi et al. 1981) and luteal production of progesterone (Ladenheim et al. 1984). These findings suggest that effects due to changes in energy balance may be mediated by insulin and is consistent with studies indicating that the probability of concep- tion is reduced in cows with low postpartum blood glucose concentrations (e.g. Plym Forshell et al. 1991, Pehrson et al. 1992). However, re- sumption of ovarian activity appears to be more dependent on the duration rather than the extent of depressed glucose concentrations (Miettinen 1991). Dietary intakes of protein and energy typi- cally considered independently to simplify ra- tion formulation, can be misleading since ener- gy supply has profound effects on the efficiency of N utilisation, while amino acids can be uti- lised for gluconeogenesis or adenosine triphos- phate synthesis (Oldham 1984). Despite a lack of experimental evidence to suggest that high protein intakes have detrimental effects on ovar- ian follicular development, ovulation or oocyte fertilisation (Blanchard et al. 1990, Garcia-Bo- jalil et al. 1994) interactions between protein and energy supply have been implicated in cases of impaired embryo development (Butler 1998). Although the relative significance of both ener- gy and protein intake and their interactions re- main unresolved, deficiencies in energy intake during periods of excessive protein feeding do appear to increase associated risks of reproduc- tive inefficiency. For example, Howard et al. (1987) reported no differences in reproductive efficiency of cows fed moderate (145 g/kg DM) or high CP (194 g/kg DM) diets. In their study, Butler and Elrod (1991) fed diets supplying pro- portionately 0.7 of calculated energy require- ments and reported that increases in dietary CP concentrations from 150 to 210 g /kg DM, were associated with a decline in conception rate from 83 to 62%. Mechanisms by which energy balance induc- es changes in the activity of the hypothalamo- hypophyseal-ovarian axis remain unresolved (Canfield et al. 1990). Since changes in endo- crine function in cows fed energy deficient diets are similar to those reported in cows fed high CP diets, effects on reproduction attributed to protein feeding could reflect changes in energy status (Ferguson and Chalupa 1989). It is cru- cial that future experimentation, examining re- lationships between protein feeding and repro- ductive efficiency effectively account for varia- tions in energy balance during early lactation. Experimental design Discrepancies in conception rate responses to changes in dietary CP content may to some ex- tent be explained by between-study differences in experimental design and subsequent analysis of experimental data. Reproductive responses may be biased or invalid if reproductive man- agement protocols, or sire and dam factors are not balanced across protein feeding treatments within experiments. Ferguson and Chalupa (1989) suggested that variations in management and sire factors within a study could be more effectively taken into account than dam factors. Experimental animals used in reproduction stud- 374 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Shingfield, K.J. et al. Protein feeding and reproductive efficiency ies are typically assigned to protein treatments based on previous milk production, parity, con- dition score and in some cases previous repro- ductive history. Since health disorders such as uterine infection (metritis) and ovarian cysts are major causes of reproductive inefficiency (Olte- nacu et al. 1984), it is essential that cows with a history of such conditions are equally distribut- ed between treatments in order to minimise bias due to dam. Reproductive responses may also be misleading due to interactions between bio- logical and management factors and variations in energy intake or energy balance. Use of sufficient experimental animals per protein treatment is also vital to allow an accu- rate assessment of associations between protein feeding and reproductive efficiency. The number of animals per treatment required is dependent on the homogeneity and reproductive potential of the sample population and the extent of dif- ferential reproductive responses expected to be identified. Ferguson and Chalupa (1989) report- ed that a minimum of between 20 to 25 and be- tween 80 and 100 cows per treatment would be required to statistically validate differential con- ception rate responses of 20 and 10%, respec- tively. Clearly, many studies have used insuffi- cient numbers of cows per treatment to allow a reliable assessment of the influence of dietary CP content on reproductive efficiency. Variations in energy intake and energy bal- ance between-studies are often impossible to reconcile, simply because energy contents of experimental diets are in many cases not report- ed. Furthermore, mobilisation of energy reserves appears to be dependent on absorbed amino acid supply. Whitelaw et al. (1987) demonstrated that abomasal infusions of casein stimulated the mobilisation of body fat stores commensurate with an increase in milk yield. Improvements in milk production continued until N equilibrium was achieved after which catabolism of excess protein resulted in a change of carbohydrate and endocrine status causing a repartitioning of nu- trients towards body tissues rather than milk syn- thesis. Inefficient utilisation of additional die- tary protein can increase tissue urea concentra- tions (Whitelaw et al. 1987, Metcalf et al. 1996), alter the composition of uterine fluid (Jordan et al. 1983) and reduce intrauterine pH (Elrod and Butler 1993, Elrod et al. 1993). Consequently, the impact of increases in protein feeding on re- productive efficiency may potentially be related to marginal milk protein yield responses. Fol- man et al. (1981) noted that increases in dietary CP content of 40 g/kg DM, reduced conception rate, increased days open and decreased milk protein synthesis. Piatkowski et al. (1981) re- ported that increases in dietary CP concentra- tions of 46 g/kg DM caused a decrease in con- ception rate of 51%, and was associated with a marginal milk protein yield response of 0.063 g g-1. In a similar study, Bruckental et al. (1989) evaluated the influence of increases of 40 g CP/ kg DM following dietary supplementation with either soya bean or fish meal on reproductive performance. Compared to the basal diet (48%), inclusion of soya bean and fish meal were asso- ciated with mean conception rates of 43 and 52%, respectively. The magnitude of conception rate responses to increased protein intake derived from soya bean meal and fish meal were con- sistent with the extent of marginal milk protein yield responses (0.056 and 0.180 g g-1, respec- tively). Ferguson and Chalupa (1989) evaluated con- ception rate responses to changes in dietary CP content using logistic regression analysis. Based on this approach, data collected from 7 studies indicated that differential conception rate re- sponses could be better explained by variations in rumen degradable protein intake than differ- ences due to dam or reproductive management factors. Furthermore, predicted conception rates were found to be independent of dietary CP con- tent, but decreased with increased rumen degra- dable protein intake. Relationships between re- productive efficiency and protein feeding may also to be confounded by variations in uterine health, age, parity, energy and the intake of un- degraded protein. Ferguson and Chalupa (1989) concluded that changes in dietary CP content per se may only elicit minor and often inconsistent effects on conception rate, but adverse effects 375 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 8 (1999): 365–392. may occur in relatively old cows or those expe- riencing post-calving complications. When at- tempting to resolve discrepancies in reproduc- tive responses to changes in dietary CP content, it appears that the age of the cow must be taken into account. Bruckental et al. (1989) demon- strated that increases in dietary CP content from 170 to 216 g/kg DM caused a delay in the onset of first oestrus and decrease pregnancy rate in cows in their fourth or later lactation. Potential negative effects of excessive protein feeding on reproductive performance Toxicity and physiological impairment of reproductive tissues Ammonia produced from microbial degradation of dietary CP is removed from the rumen as the result of incorporation into microbial protein, absorption through the rumen wall and outflow via rumen fluid (Bodeker et al. 1990). Since, free ammonia or ammonium ions are toxic to mam- malian cells (Visek 1984) potential toxicity is prevented by conversion to urea in the liver. However, hepatic ureogenesis is limited (Sy- monds 1981, Choung et al. 1990) and once ex- ceeded concentrations of ammonia in peripheral blood can approach or exceed toxic levels. High blood ammonia concentrations have been suggested to cause a suppression of the immune system (Carroll et al. 1988). Klucinski and Targowski (1984) found that sub-toxic con- centrations of ammonia (80–160 µmol/l) de- pressed bovine lymphocyte responses to mi- togens. Reduced immune function has been sug- gested to account for increased metritis in cows fed high (200 g/kg DM) compared to low CP (130 g/kg DM) diets due to a delay in the clear- ance of uterine contaminants (Anderson and Barton 1987; ref. Ferguson and Chalupa 1989). Jordan et al. (1983) noted that feeding a low or high CP diet (120 vs. 230 g/kg DM) caused sig- nificant differences in plasma ammonia concen- tration (105 vs. 133 µmol/l). Potential detrimen- tal effects on reproductive efficiency attributed to ammonia toxicity could be misleading how- ever, since analytical procedures do not distin- guish between non-ionic (NH 3 ) and ionic (NH 4 +) forms of ammonia (Visek 1968). Despite being relatively low at physiological pH, non-ionic ammonia concentrations (0.16 and 0.25% of to- tal ammonia, Jacques et al. 1959) are primarily responsible for the toxic effects of ammonia. As a result, biologically significant changes in plas- ma ammonia concentrations may not be revealed by routine determinations (Visek 1984). Feeding high CP diets can result in an in- crease in urea concentrations in the bovine re- productive tract (Jordan et al. 1983, Duby et al. 1984, Carroll et al. 1988) and elevate vaginal and uterine ammonia concentrations (Jordan et al. 1983). In vitro studies have demonstrated that aqueous solutions of urea (range of 0.006 to 6.0% (w/w)) inhibited motility and at high concentra- tions lead to the mortality of rat spermatozoa (Dasgupta et al. 1971). Motility responses to urea were found to be dose dependent. In the same study, exposure of rabbit ovum to low urea con- centrations immediately caused this tissue to contract and darken. Higher concentrations (0.6 and 6.0%) induced severe changes in ovum struc- ture causing it to become solid and opaque. In vitro studies have also shown that urea can elic- it detrimental effects on rabbit embryos (Duby and Trischler 1986, ref. Canfield et al. 1990) a finding consistent with in vivo studies of rat embryos (Saitoh and Takahashi 1977). In con- trast, Williams et al. (1987), observed no physi- ological changes in bull spermatozoa, mouse spermatozoa or developing mouse embryos cul- tured with uterine flushings collected on d 1, 5 and 10 of the oestrus cycle of cows fed low and high CP diets (120 and 230 g/kg DM, respec- tively). Studies in non-lactating cows in posi- tive energy balance have also shown that ovari- an follicular growth and embryonic survival were unaffected by large differences (123 vs. 274 g/ 376 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Shingfield, K.J. et al. Protein feeding and reproductive efficiency kg DM) in dietary CP concentration (Garcia- Bojalil et al. 1994). Carroll et al. (1988) observed a marked increase in the urea concentration of vaginal mucus from 2.9 to 7.5 mmol/l stimulat- ed by an increase in dietary CP content of 70 g/ kg DM. Despite these increases, no effects on calving to first oestrus, days to first service, days open or number of services per conception were observed. Maintaining the environment within the uter- ine lumen is a major determinant of embryo vi- ability during early pregnancy. Conditions within the uterine lumen are subject to cyclic changes as a result of modified endometrial secretion occurring under hormonal regulation or that in- duced by the presence of a viable blastocyst (McRae 1984). Feeding high levels of CP has been reported to decrease reproductive efficien- cy, without affecting oestrus (Kaim et al. 1983, Canfield et al. 1990), suggesting that declines in conception rate may be related to changes in uterine environment. Feeding diets of different CP content has been shown to alter uterine se- cretion of urea, magnesium, potassium, phospho- rus and zinc (Jordan et al. 1983). Furthermore, the ionic composition of secretions in the repro- ductive tract is known to influence the viability of spermatozoa, ovum and zygote via direct ef- fects on cell metabolism (Hurley and Mutch 1973, Hurley et al. 1976). Consequently, impair- ments of uterine physiology could potentially explain sub-optimal conception rates in cows fed excessive protein. This suggestion is tentatively supported by studies in non-lactating heifers demonstrating that increases in dietary CP con- centrations from 155 g/kg DM to 218 g/kg DM had no affect on uterine pH at the onset of oe- strus, but by d 7 post-oestrus pH was decreased (Elrod and Butler 1993). Repeating the experi- ment with lactating cows also demonstrated that excess protein regardless of source or rumen degradability had no effect on blood, saliva, urine or intrauterine pH at oestrus, but by 7 d post-oestrus uterine pH was significantly lower in high-protein groups. A more detailed investigation of uterine func- tion has recently been made possible by devel- opment of an endometrial cell culture system allowing a pH gradient to be established between apical and basal cellular compartments (Gilbert et al. 1996). The pH gradient has been shown to be sensitive to the presence of progesterone and oestradiol, while inclusion of both hormones results in a suppression of prostaglandin PGF 2 α and PGE 2 secretion. However, inclusion of urea caused a significant reduction in the efficacy of progesterone to maintain a pH gradient between apical and basal compartments and a significant increase in prostaglandin PGF 2 α and PGE 2 se- cretion. Increases in prostaglandin PGF 2 α secre- tion are particularly significant due to their rec- ognised antagonistic effects on embryo devel- opment (Mauer and Beier 1976) and survival (Schrick et al. 1993). Changes in endocrine function Progesterone is known to have a major impact on reproductive efficiency. Follicular maturation, passage of fertilised embryos into the uterus, uterine secretions and maintenance of uterine environment necessary for pregnancy are all under the hormonal control of progesterone (Smith 1986). It has been suggested that high local or systemic urea concentrations may de- press the binding of luteinizing hormone to luteal receptors which would cause progesterone con- centrations to fall and lead to depressions in re- productive performance (Jordan and Swanson 1979a, Jordan et al. 1983). Often high circulat- ing concentrations of progesterone prior to in- semination have been observed to be associated with higher conception rates (Folman et al. 1973, Erb et al. 1976, Fonseca et al. 1983) while other studies have not identified a positive influence of progesterone (Bulman and Lamming 1978, Carroll et al. 1988). Feeding high levels of pro- tein have been reported to lower plasma proges- terone concentrations (Jordan and Swanson 1979b, Carroll et al. 1988, Sonderman and Lar- son 1989). In contrast, alterations of plasma luteinizing hormone or progesterone profiles have not been identified following excessive 377 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 8 (1999): 365–392. protein feeding to non-lactating cows (Blau- wiekel et al. 1986) or beef cattle (Rusche et al. 1993). Discrepancies between these studies may be explained by variations in the extent of pro- tein degradation in the rumen or differences in magnitude of negative energy balance. Recent- ly, Garcia-Bojalil et al. (1998) demonstrated that both peak and accumulated plasma progesterone concentrations assessed during the first 50 d of lactation were significantly related to dietary rumen degradable protein content. Diets were formulated to provide similar amounts of CP (206 and 207 g/kg DM) but different amounts of rumen degradable protein (111 and 157 g/kg DM, respectively). Increases in daily dietary rumen degradable protein intake from 2.18 to 3.05 kg were associated with large differences in peak (18 and 26 µmol/l, respectively) and accumula- tive progesterone concentrations (1.7 and 2.9 mmol/l, respectively). Responses were also as- sociated with changes in dietary energy content achieved by inclusion (22 g/kg DM) of rumen protected fat. Inclusion of fat lead to increases in both peak (30 vs. 25 µmol/l) and accumula- tive progesterone concentrations (2.7 vs. 1.9 mmol/l). Over the course of three cycles during early lactation, plasma progesterone concentrations tend to increase, but the rate and extent of in- crease appears to be modified by the extent of negative energy balance (Villa-Godoy et al. 1988, Spicer et al. 1990). Energy deficiencies can lead to depressed conception rates (e.g. Park- er and Blowey 1976, Youdan and King 1977), which may be attributed to reduced plasma pro- gesterone concentrations due to a lowered sen- sitivity of luteal cells to luteinizing hormone (Apgar et al. 1975). Decreased plasma proges- terone concentration during early lactation lead- ing to compromised uterine function appears to be the most probable cause of impaired fertility in cows fed excessive protein. Relationships between reproductive efficiency and protein feeding may often be confounded by variations in energy status since both protein and energy supply appear to impinge on the hypothalamo- hypophyseal-ovarian axis, resulting in modified luteal production of progesterone (Ferguson and Chalupa 1989). Based on studies conducted with dairy cows there is little evidence to suggest that excessive protein feeding causes detrimental ef- fects on follicular maturation, ovulation or fer- tilisation, but the most convincing experimental evidence suggests an associated reduction in em- bryo survival due to a sub-optimal uterine envi- ronment (Butler 1998). Associations between protein feeding and reproductive efficiency in Finland Data concerning the association between repro- ductive efficiency and protein feeding under Finnish production situations is limited. Repro- ductive studies in Finnish cows have tended to concentrate on the role of energy intake (Miet- tinen 1990a, 1991), feeding regimen (Heinonen et al. 1988, Miettinen 1990c) breed (Kuni and Pirinen, 1988) or parity (Miettinen 1990b) on reproductive efficiency. In Finland, dairy cow rations contain only relatively moderate CP concentrations, typical- ly 150 g/kg DM (Kaustell et al. 1998) suggest- ing that potential reproductive inefficiencies due to excessive protein feeding are likely to be min- imised. Furthermore, Miettinen (1991) reported that the proportion of cows fed diets according to Finnish feeding standards with plasma urea concentrations lower than 150 mg/l at 14 and 60 d postpartum was 0.89 and 0.65, respectively. Plasma urea concentrations of cows above this threshold had much higher conception rates (71%) compared to cows with values below this concentration (52%). About 60% of Finnish dairy herds participate in a national milk recording scheme and account for approximately 73% of total milk production in Finland. In 1993, 20 018 herds participated in this scheme. Recording was initiated when cows 378 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Shingfield, K.J. et al. Protein feeding and reproductive efficiency were turned out to pasture during May 1993 and was continued to the end of the indoor housing period the following year. Milk production of recorded herds was measured monthly and milk fat, protein, lactose and urea content were de- termined bimonthly (Karjantarkkailutulokset 1993). On-farm consumption of feeds was esti- mated based on assessments of feed stores in the autumn, and was reported in the beginning, mid- dle and at the end of the indoor housing period. The contribution of grazed grass was estimated as the difference between annual energy require- ments predicted according to milk production and recorded consumption during the indoor housing period. Total consumption of feeds by dairy cows fed according to Finnish feeding standards adopted in 1993 (Salo et al. 1990) was divided by the number of cows in a herd to give an estimate of the mean annual intake of dairy cows within a herd. More detailed information concerning feed evaluation and sampling proce- dures used to obtain this data are documented by Kaustell et al. (1998). Based on the criteria that records of milk composition, feed consump- tion and reproductive parameters of recorded herds should be complete, associations between on-farm protein feeding and reproductive effi- ciency were examined using measurements from 16 051 herds (refer to Table 2). Use of field data to assess associations be- tween on-farm protein feeding and reproductive efficiency is subject to criticism, because auto- correlation between nutritional parameters and confounding effects due to standard feeding pol- icies are not completely taken into account. Ef- fects due to cow age (Ferguson and Chalupa 1989) or reproduction management (Barton et al. 1996) will also introduce additional bias, since failure to detect oestrus for example has been suggested to be the main reason for extend- ed calving intervals (Whitmore 1984). Account- ing for differences in on-farm management is particularly problematic, because highly produc- tive herds are likely to operate under the most Table 2. Summary of intake, production and reproductive efficiency data derived from 16 051 Finnish dairy herds participating in the national recording scheme during 1993, used to examine the association between on-farm protein feeding and reproductive efficiency in Finnish dairy herds. Mean SD Min Max Number of cows per herd 14 5.7 1 221 Milk production and composition Milk yield (kg/cow per year) 6719 936.6 2789 11853 Fat (g/kg) 44.3 3.7 29.1 66.9 Protein (g/kg) 32.8 1.1 28.6 39.9 Urea (mg/l) † 250 46.3 15 432 Intake (/cow per year) Dry matter (kg) 5679 658.2 3367 9041 Metabolisable energy (MJ) 63321 7634 35685 106447 Crude protein (kg) 853 117.5 445 1986 Diet composition (g/kg dry matter) Crude protein 150 10.3 83 226 Forage 670 67 250 910 Concentrate 330 67 90 750 Reproductive efficiency Calving interval (d) 387 21.8 320 605 First service interval (d) 80 14.1 23 189 Inseminations per calving 1.7 0.38 1 5 † Determined in 6 731 recorded herds 379 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 8 (1999): 365–392. effective management regimens. Furthermore, assessment of protein feeding based entirely on dietary CP content does not account for varia- tions in rumen degradable protein intake. Accept- ing these criticisms and a lack of alternative data, evaluation of field data was conducted by class- ing measurements of nutrient intake, milk pro- duction and reproductive efficiency according to dietary CP content. Classification was performed over the entire range of dietary CP concentra- tions in increments of 5 g CP/kg DM, generat- ing 17 protein classes. Use of mean values of each protein class (range 114–205 g/kg DM) indicated that annual milk production was close- ly related to dietary CP content (Fig. 2). Based on this relationship, optimal milk production was associated with feeding diets containing between 160 and 180 g CP/kg DM. However, marginal milk yield responses to increases in dietary CP content between 170–180 g/kg DM are unlikely to be sufficient to cover additional feed- ing costs. Use of the same approach tended to indicate that reproductive parameters were optimised at different dietary CP contents necessary for high- est milk production (Fig. 3). Measures of repro- ductive efficiency, i.e. calving interval, first serv- ice interval and number services per calving ap- peared to be most efficient in herds fed diets containing between 155–160, 165–170 and 130– 135 g CP/kg DM, respectively. Feeding diets containing insufficient or excessive protein tend- ed to result in an extended calving interval, a prolonged first service interval and an increase in the number of services per calving. The ex- tent of reproductive inefficiency appeared to be marginally greater for herds fed low compared to high levels of dietary protein. In herds (n = 151) fed diets containing less than 125 g CP/kg DM, calving interval, first service interval and number of services per calving were on average 394, 84 and 1.73, respectively. Mean calving interval, first service interval and number of services per calving were 392, 80 and 1.84, re- spectively in herds (n = 21) fed diets containing levels of CP in excess of 190 g/kg DM. Due to high concentrate costs and a shortage of home grown protein supplements, Finnish milk production has been reliant on feeding high quality forages. During 1993, protein evaluation in Finland was based on digestible crude pro- tein (ARC 1965, Salo et al. 1990) which cou- pled with restrictions in the availability of pro- tein supplements lead to the use of increased ni- trogen fertiliser application to increase grass CP content. Due to rapid and extensive degradation Fig. 2. Influence of dietary crude protein content on annual milk production of Finnish milk record- ed herds during 1993 (n = 16 051) based on classification according to dietary crude protein content. Each point represents mean (+ SE) of herds in each class and the dotted line indicates the mean of all recorded herds. 380 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Shingfield, K.J. et al. Protein feeding and reproductive efficiency of silage protein (Huhtanen 1998a), a close re- lationship between silage CP content and repro- ductive efficiency of Finnish herds may be ex- pected. Examination of data from recorded herds tended to indicate that associations between re- productive efficiency and silage CP content were consistent with and only marginally better than that based on dietary CP content (data not pre- sented). Examination of field data indicated that im- plementation of on-farm feeding strategies to increase dietary CP content to approximately 170 g/kg DM would lead to improved milk produc- tion equivalent to 500 kg/cow per year. Repro- ductive efficiency of recorded herds fed diets containing 150 g CP/kg DM was defined by a mean 387 d calving interval, 80 d first service interval and 1.74 inseminations per calving. Corresponding values in herds fed diets contain- ing 170 g CP/kg DM were 387, 79 and 1.77, re- Fig. 3. Association between dietary crude protein content and reproductive efficiency of Finnish milk recorded herds during 1993 (n = 16051) based on classification according to dietary crude protein content. Each point represents mean (+ SE) of herds in each class and the dotted line indicates the mean of all recorded herds. spectively. If the limitations of field data and the lack of rigorous statistical evaluation are accept- ed, measurements derived from Finnish record- ed herds tentatively supports the suggestion that increases in dietary CP content from 150 to 170 g/kg DM would not impose large constraints on reproductive efficiency. Monitoring on-farm protein feeding Due to variations in chemical composition of feed ingredients and errors inherent in all pro- tein evaluation systems, inaccuracies in dairy cow protein feeding can occur, which can lead to an inefficient utilisation of dietary protein. It 381 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 8 (1999): 365–392. is well established that measurements of urea concentrations in blood often reflect the efficien- cy of utilisation of dietary CP (e.g. Lewis 1957). Numerous studies have been conducted over the last two decades to assess the potential of urea concentrations in blood or milk as a diagnostic test of N utilisation in the dairy cow (refer to DePeters and Ferguson 1992, Shingfield et al. 1997). Recent evaluations have demonstrated that urea concentrations in milk are closely re- lated to the proportion of dietary protein degrad- ed in the rumen (Schepers and Meijer 1998), the ratio of dietary CP to energy intake or dietary CP content (Broderick and Clayton 1997, Hof et al. 1997). Examination of data derived from 18 Finnish production trials has also indicated that the ratio of CP to energy intake is the major nu- tritional factor affecting the urea concentration in milk (Shingfield and Huhtanen 1998). Associations between urea concentrations in blood and milk with reproductive efficiency Plasma urea concentration A number of reproductive studies have docu- mented an association between plasma urea con- centration (PUC) and reproductive performance. Kaim et al. (1983) reported that pregnancy rates were lower in cows with a PUC of 360 mg/l com- pared to cows with a PUC of 193 mg/l. A urea concentration in blood exceeding 429 mg/l has also been reported to be associated with reduced conception rates (Ferguson et al. 1988, 1993). Collection of blood samples from 160 cows on the day of insemination indicated that protein feeding regimens leading to PUC values above 407 mg/l were associated with a 20 percentage point decrease in pregnancy rate (Butler et al. 1996). In order to examine associations between PUC and reproductive efficiency assessed as conception rate, data from 10 published studies were compared. Measurements of urea reported in whole blood were recalculated as their con- centration in plasma according to Broderick and Clayton (1997) where PUC (mg/l) = 1.021 x urea concentration in whole blood (mg/l) + 8.55 (n = 226, r2 = 0.918). Increases in PUC were associ- ated with depressions in conception rate in sev- eral, but not in all cases (Fig. 4). In some stud- ies increases in PUC elicited positive concep- tion rate responses (Miettinen 1991, Barton et al. 1996). Discrepancies between conception rate responses to variations in PUC may be related to between-study differences in experimental and management factors or blood sampling proto- cols, since PUC is subject to considerable diur- nal variation (Miettinen and Juvonen 1990, Gus- tafsson and Palmquist 1993). In conclusion, ex- amination of data from 10 studies does not pro- vide convincing evidence to support suggestions that measurements of PUC would be beneficial in maintaining or improving on-farm reproduc- tive efficiency. Furthermore, implementation of a strict reproductive management regimen has been shown to result in high reproductive effi- ciencies irrespective of PUC (Barton et al. 1996). Milk urea concentration In contrast to blood, milk is regarded as a more ideal test medium since it is collected quantita- tively, easily sampled (Giesecke et al. 1994) and routinely measured on a large scale in many European countries as part of a national milk recording scheme (Emanuelson et al. 1989). Milk urea concentrations (MUC) appear to be direct- ly related to concentrations in plasma (e.g. But- ler et al. 1996, Metcalf et al. 1996) and there- fore several studies have assessed the incidence of reproductive inefficiencies in relation to MUC. Based on an evaluation of reproduction records of 915 German cows, Wenninger and Distl (1994) reported that significant curviline- ar relationships existed between MUC with days 382 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Shingfield, K.J. et al. Protein feeding and reproductive efficiency open, inseminations per service, conception rate at first service and the incidence of metritis. Based on this data, reproductive traits appeared to be optimised in cows with a MUC of between 150 and 250 mg/l. Sato et al. (1996) reported that cows with MUC in excess of 386 mg/l at the time of insemination failed to become preg- nant. However, these findings were not con- firmed by measurements in the subsequent year of the study. Gustafsson and Carlsson (1993) re- ported that intervals between calving and first (mean 82 d) and last (mean 108 d) services were delayed in herds with low or high bulk tank MUC. Calving to last service intervals were found to be much shorter in herds with a mean MUC between 270 and 300 mg/l. These find- ings are consistent with studies which have iden- tified reproductive inefficiencies in cows with either low (Pehrson et al. 1992, Carlsson and Pehrson 1993) or high MUC (Carroll et al. 1988, Ferguson et al. 1988, Canfield et al. 1990, Pehr- son et al. 1992, Butler et al. 1996). The associa- tion between MUC and reproductive efficiency has often been described by non-linear relation- ships indicating optimal reproduction efficien- cy at concentrations between 150–250 (Wennin- ger and Distl 1994), 240–410 (Pehrson et al. 1992) and 270–300 mg/l (Gustafsson and Carls- son 1993). Over the last 10 years, urea concentrations of bulk tank milk have been routinely measured to monitor protein feeding on Finnish dairy farms. Concentrations of urea in milk between 200 and 300 mg/l have been used to describe an efficient utilisation of dietary protein. On-farm protein feeding regimens resulting in bulk tank MUC within these limits are considered to be optimal with respect to both reproductive effi- ciency and milk production. During the last cou- ple of years the proportion of Finnish bulk tank milk samples with urea concentrations deter- mined using an enzymatic method (Rajamäki and Rauramaa 1984) above 300 mg/l has markedly increased (Table 3). Data from Sweden has also indicated that MUC of samples collected from individual cows were on average 300 mg/l be- tween October 1996 and July 1997, and that 39% of samples from 51–111 d postpartum cows had concentrations in excess of 324 mg/l (Eriksson and Gustafsson 1998). Since the implementation of routine meas- urements, MUC recommendations have re- mained unchanged, despite annual milk produc- tion increases of approximately 100 kg per cow over the last decade (Nousiainen 1997). It has recently been suggested that in order to realise potential improvements in milk production the range of MUC used for advisory purposes should Fig. 4. Association between plas- ma urea concentration and concep- tion rate reported in the literature. Data derived from Folman et al. 1981 (+), Piatkowski et al. 1981 (�), Kaim et al. 1983 (o), Howard et al. 1987 (+), Carroll et al. 1988 (▲), Bruckental et al. 1989 (●), Canfield et al. 1989 (�), Pehrson et al. 1992 (�), Elrod and Butler 1993 (�) and Barton et al. 1996 (�). 383 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 8 (1999): 365–392. be modified to 200–350 mg/l (Shingfield et al. 1997, Shingfield and Jokela 1998), but the po- tential impact of these changes on reproductive efficiency is unclear. Despite the large number of Finnish herds (n = 20 018) participating in the national milk recording scheme, MUC measurements of bulk tank were conducted for only 6 731 herds. Of these, 566 herds had less than 3 measurements performed during the entire recording period. Based on the criteria that estimates of the aver- age annual urea concentration in bulk tank milk should be based on the mean of a minimum of 6 bimonthly assessments, records of 5 437 herds were used to assess the association between MUC and the reproductive efficiency under Finnish conditions. Milk production, reproduc- tive efficiency and MUC data derived from these recorded herds is summarised in Table 4. Recording data was evaluated by classifying herds according to the mean annual MUC in increments of 20 mg/l, generating 17 MUC classes. Classes that contained less than 14 herds, proportionately 0.0025 of sample popu- lation were considered to be unreliable. Con- sequently, evaluation was conducted using 14 classes over a range of bulk tank MUC between 152 and 368 mg/l. Based on this approach, an- nual milk production was observed to highest in herds with a mean annual bulk tank MUC of 308 mg/l (Fig. 5). In contrast, associations be- tween bulk tank and reproductive performance of Finnish herds were less well defined. Increas- es in bulk tank MUC from 200 to 300 mg/l in Finnish herds was associated with only minor differences in calving interval (Fig. 6), a 2 d reduction in the first service interval (data not presented) and an increase (0.05) in the number of inseminations per calving (Fig. 7). Finnish herds (n = 4268) with an estimated mean annu- al bulk tank MUC within the range recommend- ed in 1993 (200–300 mg/l) had calving and first intervals of 387 (+ 2.5) and 80 (+ 2.0) d, re- spectively and required 1.7 (+ 0.06) insemina- tions per calving. Mean calving intervals, first service intervals and number of services per calving of herds (n = 616) with bulk tank MUC between 300 and 350 mg/l were 388 (+ 1.8) d, 81 (+ 2.1) d and 1.7 (+ 0.09), respectively. Cor- responding values for herds (n = 503) with an- Table 3. Urea concentrations of bulk tank milk in Finland between 1988 and 1999. Indoor housing Sample Milk urea concentration (mg/l) † Period number Mean Proportion of samples (SD) <200 200–300 >300 1988–91 1 240 000 ND 0.22 0.63 0.15 1990–91 1 51 000 ND 0.14 0.61 0.26 1991–92 1 86 000 ND 0.14 0.52 0.34 1992–93 1 95 000 ND 0.29 0.55 0.17 1993–94 1 93 000 249 (74) 0.25 0.52 0.23 1994–95 1 86 334 266 (77) 0.19 0.48 0.33 1995–96 1 140 203 252 (82) 0.21 0.43 0.35 1996–97 2 142 496 243 (77) 0.28 0.48 0.24 1997–98 2 149 424 300 (77) 0.09 0.38 0.52 1998–99 3 87 968 294 (86) 0.14 0.37 0.49 ND: Not determined † Determined according to Rajamäki and Rauramaa (1984) 1 Derived from Nousiainen (1997) 2 Derived from Tommila and Jokela (unpublished data) 3 Derived from Nousiainen (unpublished data) 384 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Shingfield, K.J. et al. Protein feeding and reproductive efficiency nual bulk tank MUC below 200 mg/l were 386 (+ 5.0) d, 83 (+ 2.3) d and 1.7 (+ 0.10), respec- tively. The means and associated standard devia- tions for these reproductive parameters were re- markably similar between herds with mean an- nual urea concentrations in bulk tank MUC of below 200 mg/l, between 200 and 300 mg/l and above 300 mg/l. These findings tentatively sug- gest no consistent relationship between on-farm Table 4. Summary of intake, production and reproductive efficiency data derived from 5 437 Finnish dairy herds participating in the national recording scheme during 1993, used to examine the association between mean annual urea concentration in bulk tank milk and reproductive efficiency in Finnish dairy herds. Mean SD Min Max Number of cows per herd 14 5.2 2 104 Milk production and composition Milk yield (kg/cow per year) 6735 935.5 3491 11136 Fat (g/kg) 44.4 3.8 29.5 66.9 Protein (g/kg) 32.8 1.1 28.6 37.5 Urea (mg/l) 254 40.7 82 411 Intake (/cow per year) Dry matter (kg) 5647 665.0 3386 9041 Metabolisable energy (MJ) Crude protein (kg) 856 115.5 448 1703 Diet composition (g/kg dry matter) Crude protein 152 9.6 115 214 Forage 666 67 253 888 Concentrate 334 67 112 747 Reproductive efficiency Calving interval (d) 386 21.2 333 605 First service interval (d) 79 13.9 42 189 Inseminations per calving 1.7 0.37 1 4.2 Fig. 5. Relationship between urea concentrations in bulk tank milk and annual milk production of Finnish milk recorded herds (n = 5 437) during 1993 based on clas- sification according to milk urea concentration. Each point repre- sents mean (+ SE) of herds in each class and the dotted line indicates the mean of all recorded herds. 385 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 8 (1999): 365–392. reproductive efficiency and mean annual bulk tank MUC. Notwithstanding the use of data col- lected from relatively large sample population (n = 5 437 herds) and MUC being determined on a minimum of 6 occasions, these findings are equivocal, since the effects due to standard feed- ing policies, cow age or reproduction manage- ment are confounded. Use of estimates of the mean annual bulk tank MUC of Finnish herds is also subject to criticism since it hides the true variation in MUC due to factors such as stage of lactation (e.g. Emanuelson et al. 1993, Carlsson et al. 1995). Furthermore, measurements of MUC in bulk tank reflect the average MUC of an indi- vidual herd (Refsdal 1983, Carlsson et al. 1995) and therefore represent an integrated mean of MUC from both pregnant and non-pregnant an- imals. In a similar evaluation based on measure- ments collected from 256 herds in Norway, dif- ferences in herd geographical location were iden- Fig. 6. Relationship between urea concentrations in bulk tank milk and calving interval of Finnish milk recorded herds during 1993 (n = 5 437) based on classification according to milk urea concentra- tion. Each point represents mean (+ SE) of herds in each class and the dotted line indicates the mean of all recorded herds. Fig. 7. Relationship between urea concentrations in bulk tank milk and number of inseminations per calving of Finnish milk recorded herds during 1993 (n = 5 437) based on classification according to milk urea concentration. Each point represents mean (+ SE) of herds in each class and the dotted line indicates the mean of all re- corded herds. 386 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Shingfield, K.J. et al. Protein feeding and reproductive efficiency tified as an important limitation to the use of bulk tank MUC as a diagnostic of protein feeding with respect to reproductive efficiency (Ropstad and Refsdal 1987). Despite these criticisms and a distinct lack of alternative information, examination of re- cording data collected during 1993 tended to indicate that implementation of recommended urea concentrations in bulk tank milk of between 200 and 350 mg/l would not impose large con- straints on the reproductive efficiency of Finn- ish dairy herds. Comparison of the reproductive efficiency of recorded herds with mean annual bulk tank urea concentrations within the range of 200–300 mg/l and 300–350 mg/l, indicated that increases in MUC may be associated with on average, a 3 d extension of calving and first service intervals. It is however impossible from evaluation of field data to accurately predict the magnitude of reproductive responses to changes in bulk tank MUC for an individual herd. Conclusions Reproductive responses of dairy cows to increas- es in dietary CP concentrations tend to be in- consistent. Most, but not all studies have report- ed a negative association between reproductive performance and increased protein feeding. Dis- crepancies in reproductive responses may arise due to between-study differences in experimen- tal design, uterine health, cow age, parity, nutri- ent intake, reproductive management or the size of sample populations. Detrimental effects on reproductive efficiency attributed to excess pro- tein feeding often appear to be more clearly as- sociated with either an excessive intake of ru- men degradable or undegradable protein and the extent of postpartum negative energy balance. The most convincing experimental evidence sug- gests that detrimental effects associated with excessive protein feeding appear to be mediated by increased concentrations of urea in peripher- al blood leading to compromised uterine func- tion. Evaluation of data collected from Finnish herds participating in the National milk record- ing scheme during 1993, indicated that feeding diets containing between 170 and 180 g CP/kg DM resulted in optimal milk production. Further- more, comparison of herds feeding diets contain- ing between 140 and 180 g CP/kg DM, suggest- ed that increased on-farm protein feeding is not consistently associated with compromised repro- ductive efficiency. 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Julkaistun aineiston perusteella valku- aisruokinnan vaikutus hedelmällisyyteen vaihtelee. Hedelmällisyyden parametreinä käytettiin tyhjäpäivi- en määrää, siemennysten määrää tiineyttä kohti ja tii- nehtymisprosenttia. Vaihtelevat tulokset voivat joh- tua erilaisista koejärjestelyistä, tilastollisista analyy- simenetelmistä, havaintojen määrästä (usein liian pie- ni hedelmällisyyden mittaamiseksi luotettavasti), kohdun terveydentilasta, lehmän iästä, poikimisker- tojen määrästä, lehmien hoidosta ja ravintoaineiden saannista. Liian runsaan valkuaisruokinnan aiheutta- maan heikentyneeseen hedelmällisyyteen liittyy usein kudosten urea- ja ammoniakkipitoisuuden nousu, mikä huonontaa lisääntymiselinten fysiologista toi- mintaa, muuttaa hormonien eritystä tai lisää negatii- vista energiatasetta poikimisen jälkeen. Vuoden 1993 tarkkailuaineistojen (16 051 karjaa) analysointi osoit- ti, että maidontuotannon optimi saavutettiin rehuan- noksen raakavalkuaispitoisuuden ollessa 180 g/kg kuiva-ainetta (ka) hedelmällisyysparametrien pysyes- sä lähes ennallaan. Tutkittaessa tankkimaidon urea- pitoisuuden vaikutusta hedelmällisyyteen 5 437 tilan aineistossa havaittiin, että tilojen väliset erot maidon ureapitoisuudessa eivät vaikuttaneet hedelmällisyy- teen. Johtopäätöksenä voidaan todeta, että rehuannok- sen raakavalkuaispitoisuuden nostaminen 150:stä 170 g:aan/kg ka lisää maidontuotantoa huonontamatta merkittävästi hedelmällisyyttä. Title Introduction Protein feeding Association between protein feeding and reproductive efficiency Potential negative effects of Associations between protein Monitoring on-farm protein Associations between urea Conclusions References SELOSTUS