2016: The use of artificial insemination gun protective plastic sheath at the time of artificial insemination did not improve fertility of beef cattle The use of artificial insemination gun protective plastic sheath at the time of artificial insemination did not improve fertility of beef cattle Ramanathan Kasimanickam Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, WA Abstract A strict hygienic artificial insemination (AI) technique is critical to maximize reproductive outcomes. The objective of this study was to evaluate the effect of AI gun protective plastic sheaths use on AI pregnancy rates (AI-PR) in beef cattle. Angus cross beef cows (n=321) and Angus cross beef heifers (n=430) were randomly assigned to one of two groups: with (TRT) or without (CON) the use of disposable plastic sheath during the AI procedure. Accounting for estrus expression at or prior to AI (P<0.01), the use of protective sheath neither in cows (sheath, 59.4% [95/160] vs. no sheath, 55.9% [90/161]; P>0.1] nor in heifers (sheath, 58.3 [127/218] vs. no sheath, 56.7% [121/212]; P>0.1) improved AI-PR. Both cows and heifers that expressed estrus at or prior to AI had greater AI-PR compared to that did not express estrus (cows: estrus, 64.4% [116/180] vs. no estrus, 48.9 [69/141]; heifers: estrus, 64.6 [144/213] vs. no estrus, 50.2 [104/207]; P<0.01). In conclusion, carrying out a hygienic AI technique is critical for AI success; however, the use of protective sheath did not improve reproductive outcomes in beef cattle. Keywords: Beef cattle, artificial insemination, AI gun, protective sheath, pregnancy rate Introduction Artificial insemination was the first reproductive biotechnology applied to improve reproduction and genetics of farm animals.1 The utilization of AI has an enormous impact worldwide in many species, mainly among domestic livestock including cattle, sheep, goats, pigs, horses, poultry (turkeys, ducks, fowl) and rabbits. The worldwide acceptance of AI technology provided a thrust for the development of other assisted reproductive technologies, such as pharmacological regulation of estrous cycle, multiple ovulation and embryo transfer, in-vitro embryo production, sexing of sperm and embryo, cryopreservation of gametes and embryo, and somatic cell nuclear transfer and cloning.1,2 In cattle the safest and best method of insemination is the recto-vaginal method which involves traversing the anatomic barriers of the female reproductive tract, the cervix, and deposition of frozen-thawed semen in the body of the uterus under hygienic conditions. The efficiency of insemination depends on the deposition of proper numbers of normal spermatozoa at the appropriate site in the reproductive tract at the precise time relative to estrus. In addition, an adequate and clean AI technique is recommended to improve reproductive outcome.3 Improper handling of instruments and unsanitary conditions may lead to lower fertility. Cattle fertility is influenced by several factors including herd environment and management practices.4 It should be noted that during the AI procedure, not only semen but also bacteria and debris may be introduced into the lumen of the uterus. Studies revealed that dairy cows that developed post-AI subclinical endometritis had lower conception rates.5 This could be due to either uterine reaction to sperm, existing infection, or introduction of pathogens from the vagina into the uterus. In beef cows endometrial cytology did not predict final pregnancy status or day of conception when samples were collected >50 d postpartum, indicating that beef cows were able to clear uterine inflammation after resumption of ovarian cyclicity.6 However, a recent study that followed beef cows for 130 days postpartum concluded that cows with subclinical endometritis had an increase in time to conception and a decrease in the risk of pregnancy.7 Use of protective sheaths (double sheath insemination procedure) prevents introduction of vaginal contaminants including pathogenic organisms into the uterus via the AI gun. Previous studies reported no improvement in first service conception rates3,8 and improved pregnancies per AI for second or greater Clinical Theriogenology • Volume 8 Number 2 • June 2016103 services in dairy cattle9 following the use of AI catheter protective sheaths during AI compared to no protective sheath use. Use of protective sheaths incurs additional cost to beef producers whereas failure to use protective sheaths may lower pregnancy rate. This study was designed with an intent to provide a comprehensive recommendation on whether or not to use AI gun protective sheaths while performing AI in beef cattle. The objective of this study was to evaluate the effect of AI gun protective plastic sheaths (PS) use on AI pregnancies in beef cattle. Materials and methods Animals and breeding management Cows. A total of 321 Angus cross beef cows from three similarly managed commercial cow-calf operations inseminated during spring of 2014 were included. Within locations cows were randomly assigned to Ovsynch+ controlled internal drug release (CIDR) or CO-Synch+CIDR groups (Figure 1A) and were assigned a body condition score (BCS; 1-emaciated; 9-obese) at initiation of synchronization protocol (Day 0). Cows in Ovsynch+CIDR group received a CIDR (1.3 g of progesterone; per vagina; Eazi-Breed™ CIDR® Cattle Insert; Zoetis Animal Health, New York, NY) vaginal insert and 100 µg gonadorelin diacetate tetrahydrate (GnRH; 2 mL; im; Cystorelin®, Merial Inc., Duluth, GA) on Day 0, 25 mg prostaglandin F2alpha (PGF; dinoprost tromethamine sterile solution, 5 mL; im; Lutalyse®, Zoetis Animal Health) and CIDR removal on Day 7, 100 μg GnRH, im, 48 h later on Day 9 and insemination on Day10, 66 hrs after CIDR removal. Cows in CO-Synch+CIDR group received treatment similar to cows in Ovsynch+CIDR group except the second GnRH (100 μg; im) was administered at the time of AI. Sires (n=5) were selected and assigned to cows based on sire traits and to avoid inbreeding. Inseminators (n=6) included in the study were experienced but differed among locations. Cows were maintained in pastoral conditions and during the winter months supplemented with hay. The cows were fed to meet NRC recommendations.10 At the time of AI, cows were randomly assigned to one of the two treatment groups, with (TRT, n = 160) or without (CON, n = 161) the use of AI catheter protective sheath. The sheath (30 cm in length × 0.7 cm in diameter) was a rigid polyvinyl chloride tube (King et al., 1984). The sheath was funnel shaped at one end, where the insemination catheter was introduced, whereas the other end was sealed with a prescored soft rubber cap that can be easily punctured when pressure was applied. For all services performed in the TRT group, the AI catheter protected with a sheath was introduced in to the vagina; at the cranial portion of the vagina adjacent to the cervical os, the sheath was punctured by withdrawing it back, exposing the AI catheter that was advanced through the cervix into the uterine body for semen deposition. In the CON group, the AI catheter was introduced to the vagina without the use of sheath and was advanced through the cervix for deposition of the semen in the uterine body. All cows were fitted with a heat detector aids estrus detection aids (Kamar® Heatmount detector patches; Kamar, Inc., Steamboat Springs, CO) or Estrus Alert patches (Western Point Inc., Apple Valley, MN) or chalk at CIDR removal. After CIDR removal, the cows were observed twice daily until insemination for estrus and estrus detection aid status (estrus, activated aids or lost aids with mount marks vs. no estrus, intact aids) and estrus status (standing to be mounted) was recorded. A cow was determined to be in estrus if she was observed to stand for mounting or if she had an activated, lost (with mount marks) or partially-activated aid. The timing of CIDR insertion, CIDR withdrawal, and interval from CIDR withdrawal to timed-AI was recorded for each animal. Age of the cows was retrieved from the records. Two weeks later, intact Angus bulls were placed with cows (approximately1:30 to 1:50), across treatments, for the remainder of the 60 to70 d breeding season. Cows were examined for pregnancy status approximately 70 days after AI by ultrasonography of the uterus and its contents to differentiate cows bred by AI or natural service.11,12 The criteria considered were the size of the amniotic vesicle, fetus, and placentomes. The AI pregnancy rate was calculated as the number of cows pregnant to AI divided by the total number of cows inseminated. Clinical Theriogenology • Volume 8 Number 2 • June 2016 104 Heifers. A total of 430 Angus cross beef heifers from four similarly managed commercial cow- calf operations inseminated during spring of 2013 were included. The goal of the participating farms was to have heifers calve at two year of age. The heifers were fed to meet NRC recommendations.10 At enrollment, heifers were assigned a BCS (1 to 9; 1, emaciated; 9, obese) and a reproductive tract score (RTS, 1 to 5; 1, immature, anestrus; 5, mature, cycling). Within the herd, heifers were randomly assigned to long term (LT)-72 (14-d CIDR-PGF-GnRH; n=203) or short term (ST)-72 (5-d CO-Synch+CIDR; n=227) estrous synchronization protocol groups (Figure 1B). Briefly, heifers in the LT-72 group received a CIDR from Days 0 to 14, followed by 25 mg of PGF 16 days later (Day 30). In order to inseminate heifers in both LT and ST groups at the same time, the synchronization of heifers in ST-72 group was initiated on Day 25. Heifers in the ST-72 group received a CIDR and 100 µg of GnRH on Day 25 followed by 25 mg of PGF at CIDR removal on Day 30 and a second dose of PGF six hours later (Day 30). Artificial insemination was performed at 72 h (Day 33) after CIDR removal. All heifers were given GnRH (100 µg, im) at the time of insemination. The sires (n=7) were selected and assigned to heifers based on sire traits and to avoid inbreeding. Inseminators (n=7) were experienced but differed among locations. At the time of AI, heifers were randomly assigned to one of the two treatment groups, with (TRT, n=218) or without (CON, n=212) the use of protective sheath as described previously. All heifers were fitted with a heat detector aids estrus detection aids (Kamar® Heatmount detector patches;Kamar, Inc., Steamboat Springs, CO or Estrus Alert patches; Western Point Inc., Apple Valley, MN) or chalk at CIDR removal. After CIDR removal, the heifers were observed twice daily until insemination for estrus and estrus detection aid status (estrus, activated or lost aids with mount marks vs. no estrus, intact aids) and estrus status (standing to be mounted) was recorded as described previously. The timing of CIDR insertion, CIDR withdrawal, interval to the second PGF injection and timed-AI was recorded for each animal. Age of the heifers was retrieved from the records. Two weeks later, intact Angus bulls were placed with heifers (approximately1:30 to 1:50), across treatments, for the remainder of the 60 to70 d breeding season. Heifers were examined for pregnancy status approximately 70 days after AI by ultrasonography of the uterus and its contents to differentiate heifers bred by AI or natural service sires.11,12 The criteria considered were the size of the amniotic vesicle, fetus, and placentomes. The AI pregnancy rate was calculated as the number of heifers pregnant to AI divided by the total number of heifers inseminated. Statistical analyses Data were analyzed with a statistical software program (SAS Version 9.4 for Windows, SAS Institute, Cary, NC). Mean BCS, age, days postpartum, the mean interval (h) CIDR insertion to CIDR withdrawal, and mean interval (h) from CIDR withdrawal to timed-AI in cows between TRT and CON groups were analyzed using one-way ANOVA (PROC GLM of SAS). Similarly differences in the mean BCS, RTS, age, the mean interval (h) from CIDR insertion to CIDR withdrawal, the mean interval (h) from first to second PGF injection and the mean interval from CIDR withdrawal to timed-AI in heifers between TRT and CON groups were analyzed using one-way ANOVA (PROC GLM of SAS). PROC GLIMMIX of SAS was used to examine the differences in AI pregnancy rate between TRT and CON groups. Variables included in the model for cows were treatments (sheath vs. no sheath), synchronization treatments, BCS categories (≤ 5 vs > 5), days postpartum categories (≤ 60 vs. > 60 days), age categories (2, 3 to 6 and > 6 yrs), estrus (expression at or prior to AI vs. no expression) and appropriate interaction. Variables included in the model for heifers were treatments (sheath vs. no sheath), synchronization treatments, BCS categories (≤ 5 vs > 5), age categories (≤ 15 vs.>15 mo), RTS (2 to 5), estrus (expression at or prior to AI vs. no expression) and appropriate interactions. In the model, AI sires, inseminators and locations were used as random effects. All main effects were retained during model reduction in the model. The ‘P’ value at 0.05 was considered significant. Results Clinical Theriogenology • Volume 8 Number 2 • June 2016105 The mean (± SEM) age, mean BCS and mean days postpartum for cows are given in Table 1. The mean age, mean body condition scores and mean days postpartum in cows did not differ between TRT and CON groups (Table 1; P>0.1). Similarly, the mean (± SEM) age, mean BCS and mean RTS for heifers are given in Table 1. The mean age, mean BCS and mean RTS in heifers did not differ between TRT and CON groups (Table 1; P>0.1). The proportion of RTS 5, 4 3, and 2 were 51.9 (223), 21.6 (93), 17.2 (74) and 9.3% (40), respectively. The mean time (h) from CIDR insertion to CIDR withdrawal, and interval from CIDR withdrawal to timed-AI in cows did not differ between TRT and CON groups (P>0.1). Similarly, the mean time (h) from CIDR insertion to CIDR withdrawal, interval from first to second PGF injection and time from CIDR withdrawal to timed-AI in heifers did not differ between TRT and CON groups (P>0.1). Accounting for estrus expression at or prior to AI (P<0.01), the use of protective sheath did not improve AI-PR in cows (sheath, 59.4 [95/160] vs. no sheath, 55.9% [90/161]; P>0.1; Figure 2). The AI- PR for synchronization treatments (Ovsynch+CIDR, 58.8% [100/170] vs.CO-Synch+CIDR, 56.3% [85/151]), BCS categories (≤ 5, 57.4% [58/101] vs. > 5, 57.7% [127/220]), days postpartum categories (≤ 60, 56.7% [148/261] vs. > 60 days, 61.2% [27/60]) and age categories (2, 52.0% [13/25]; 3 to 6, 59.2% [115/192] and > 6 yrs, 54.8% [57/104]), were not significantly different (P>0.1;Table 2). Cows that expressed estrus at or prior to AI had greater AI-PR compared cows that did not express estrus (estrus, 64.4% [116/180] vs. no estrus, 48.9% [69/141]; P<0.01). Accounting for estrus expression at or prior to AI (P<0.01), the use of protective sheath did not improve AI-PR in heifers (sheath, 58.3% [127/218] vs. no sheath, 56.7% [121/212]; P>0.1; Figure 2). The AI-PR for synchronization treatments (ST-72, 58.6% [133/227] vs.LT-72, 56.3% [115/203]), BCS categories (≤ 5, 57.1% [93/163] vs. > 5, 58.1% [155/267]), and RTS categories (5, 60.9% [136/223], 4, 56.5% [52/93], 3, 55.4% [41/74], 2, 47.5% [19/40]) were not significantly different (P>0.1;Table 2). Age categories had a trend for differences in AI-PR (≤ 15, 55.4% [(56/101]; > 15 mo, 58.4% [192/329]; P<0.1). Heifers that expressed estrus at or prior to AI had greater AI-PR compared heifers that did not express estrus (estrus, 64.6% [144/213] vs. no estrus, 50.2% [104/207]; P<0.01). Discussion The results of the present study revealed that the use of protective sheath did not improve AI-PR in beef heifers and in beef cows. Overall AI-PR was comparable to studies that used similar reproductive protocols.13,14 The purpose of using protective sheath is to minimize contamination of the AI catheter at the time of AI in order to prevent introduction of pathogens into the uterus and to improve reproductive outcome. Although bacterial isolation was not performed in this study, a study in dairy cattle showed a greater proportion of recognized potential and opportunistic uterine pathogens isolated from samples taken from the AI gun in cows inseminated without the protective sheath compared with cows inseminated with the protective sheath.9 The recognized uterine pathogens isolated were Trueperella pyogenes, Prevotella melaninogenica, Escherichia coli, Fusobacterium necrophorum, and Proteus spp., potential uterine pathogens isolated were Bacillus spp. and Pasteurella spp., and opportunistic uterine contaminants isolated were Streptococcus spp., Providencia spp., Klebsiella spp., and Corynebacterium spp. These pathogens have been associated with postpartum uterine diseases and reduced fertility in dairy cattle. Even though, the prevalence and severity of uterine disease in beef cattle is not as great as those in dairy cattle (17% vs. 51% prevalence at 7 wk. postpartum, respectively),6,15 it is possible that these pathogens could reside in the vagina of beef cattle. Beef females included in this study were synchronized by CIDR protocols that required vaginal placement of inserts from 5 to 14 days. The placement of an intravaginal device provides the opportunity for bacterial vaginitis. Bacterial culture of swabs of the vagina after a 7-d treatment period revealed moderate growth of coliforms, environmental Streptococcus spp. and Staphylococcus spp., and other gram-positive, rod-shaped organisms.16 Bulman et al sampled close to the cervical os after 14 d of progesterone-releasing intravaginal device (PRID) treatment and isolated Enterococcus spp., Escherichia coli, and Proteus spp. from 90% of the treated animals and 40% of animals examined with a speculum.17 Clinical Theriogenology • Volume 8 Number 2 • June 2016 106 However, from repeated sampling, the authors reported no bacterial growth in 90% of the cows 7 d after device removal and in 100% of the cows 14 d after device removal.17 It should be noted that application of vaginal insert did not reduce reproductive outcome in beef cattle. In general, beef females are exposed to bulls during the rest of the breeding season after the first AI. This poses a greater risk of beef females acquiring pathogens from beef bulls. Female reproductive tract contamination by Ureasplasma spp., Mycoplasma spp.18 and venereal pathogens such as Campylobacter fetus19,20 and Tritrichomonas fetus21 poses a greater risk for reduced reproductive outcomes. In lactating dairy cows, a higher proportion of polymorphonuclear neutrophils (PMN) (>15%) immediately before22 and 4 h after AI5 were associated with poor reproductive performance. This indicates that the dairy cows with lowered reproductive performance possibly suffered from poor uterine clearance and poor immunological response due to reduced estradiol concentration around the time of AI.23,24 It should be noted that beef cows are able to clear uterine inflammation after resumption of ovarian cyclicity.6 Beef cows that exhibited estrus had significantly greater preovulatory peak estradiol concentrations than cows not exhibiting estrus.20 Similarly, preovulatory plasma estradiol concentrations on day of AI for dairy cows diagnosed pregnant was greater compared to cows that are not pregnant.25 Results from these studies indicated that preovulatory estradiol concentrations were greater in beef cows compared to dairy cows. This elevated estradiol concentrations in beef females could possibly contributed to the required uterine clearance and immunological response around the time of AI resulted in similar AI pregnancy between TRT and CON groups. Beef females that expressed estrus had greater AI pregnancy compared to those that did not express estrus. Preovulatory estradiol is essential for preovulatory luteinizing hormone secretion,26 and preparation of ideal oviduct and uterine environments for proper embryo development.27,28 Therefore it is possible that elevated concentrations of estrogen in cows that expressed estrus had effects on follicle development and ovulation, and effects on uterine environment to provide a more conducive environment for fertilization and subsequent embryo development. Conclusions In conclusion, a hygienic AI technique is critical for AI success; however, the use of protective sheath did not improve reproductive outcomes in beef cattle. Beef females that expressed estrus had greater AI pregnancies likely as a result of differences follicle development and favorable uterine environment for fertilization and subsequent embryo development. Acknowledgements The author thanks participating beef cattle producers for the successful completion of this study. The author acknowledges Drs. Matthew Asay and Philip Firth for their assistance with this project and thank Zoetis Animal Health for donation of Eazi-Breed® CIDR Cattle Insert, Lutalyse®, and Factrel® for the heifer experiment. References 1. Foote RH: Review: dairy cattle reproductive physiology research and management-past progress and future prospects. J Dairy Sci 1996;79:980-990. 2. Vishwanath R: Artificial insemination: the state of the art. Theriogenology 2003;59:571-584. 3. King GJ, Bellissimo DJ, Penner WJ: Routine use of protective sheaths in cattle inseminations did not improve fertility. Can Vet J 1984;25:327-328. 4. Windig JJ, Calus MP, Veerkamp RF: Influence of herd environment on health and fertility and their relationship with milk production. J Dairy Sci 2005;88:335-347. 5. Kaufmann TB, Drillich M, Tenhagen BA, et al: Prevalence of bovine subclinical endometritis 4 h after insemination and its effects on first service conception rate. Theriogenology 2009;71:385-391. 6. Santos NR, Lamb GC, Brown DR, et al: Postpartum endometrial cytology in beef cows. Theriogenology 2009;71:739- 745. 7. Ricci A, Gallo S, Molinaro F, et al: Evaluation of subclinical endometritis and consequences on fertility in piedmontese beef cows. Reprod Domest Anim 2015;50:142-148. Clinical Theriogenology • Volume 8 Number 2 • June 2016107 8. Richards MW, Spitzer JC, Newman SK, et al: Bovine pregnancy and non-return rates following artificial insemination using a covered sheath. Theriogenology 1984;21:949-957. 9. Bas S, Hoet A, Rajala-Schultz P, et al: The use of plastic cover sheaths at the time of artificial insemination improved fertility of lactating dairy cows. J Dairy Sci 2011;94:793-799. 10. NRC: Nutrient requirements of beef cattle. Seventh edition. Washington, DC: Natl Acad Press; 2000. 11. Hughes EA, Davies DAR: Practical uses of ultrasound in early pregnancy in cattle. Vet Rec 1989;124:456-458. 12. Ginther OJ: Ultrasonic imaging and animal reproduction: fundamentals book 1. Cross Plains(WI): Equiservices Publishing; 1995. p. 7- 82 and 147-155. 13. Kasimanickam R, Schroeder S, Hall JB, et al: Fertility after implementation of long- and short-term progesterone-based ovulation synchronization protocols for fixed-time artificial insemination in beef heifers. Theriogenology 2015;83:1226-1232. 14. Kasimanickam R, Hall JB, Currin JF, et al: Sire effect on the pregnancy outcome in beef cows synchronized with progesterone based Ovsynch and CO-Synch protocols. Anim Reprod Sci 2008;104:1-8. 15. Gilbert RO, Shin ST, Frajblat M, et al: The incidence of endometritis and its effect on reproductive performance of dairy cows. In: Proc 12th Int Conf Prod Dis Farm Anim, 2004;12:30 [abstract]. 16. Walsh RJ, LeBlanc SJ, Bernooy E, et al: Safety of a progesterone-releasing intravaginal device as assessed from vaginal mucosal integrity and indicators of systemic inflammation in postpartum dairy cows. Can J Vet Res 2008;72:43-49. 17. Bulman DC, McKibbin PE, Appleyard WT, et al: Effect of a progesterone releasing intravaginal device on mik progesterone levels, vaginal flora, milk yield and fertility of cyclic and non-cyclic dairy cows. J Reprod Fertil 1978;53:289-296. 18. Mulira GL, Saunders JR, Barth AD: Isolation of Ureaplasma diversum and mycoplasmas from genital tracts of beef and dairy cattle in Saskatchewan. Can Vet J 1992;33:46-49. 19. Schurig GD, Hall CE, Burda K, et al: Infection patterns in heifers following cervicovaginal or intrauterine instillation of Campylobacter (Vibrio) fetus veneralis. Cornell Vet 1974;64:533-548. 20. Truyers I, Luke T, Wilson D, et al: Diagnosis and management of venereal campylobacteriosis in beef cattle. BMC Vet Res 2014;10:280. 21. Corbeil LB, Hodgson JL, Jones DW, et al: Adherence of Tritrichomonas foetus to bovine vaginal epithelial cells. Infect Immun 1989;57:2158-2165. 22. Williams BL, Senger PL, Stephens LR, et al: Relationships between days post partum, observed estrus and uterine microflora in commercial dairy cows. Theriogenology 1988:30:555-561. 23. Lacetera N, Scalia D, Franci O, et al: Short communication: effects of nonesterified fatty acids on lymphocyte function in dairy heifers. J. Dairy Sci 2004;87:1012-1014. 24. Leroy JLMR, Vanholder T, Delanghe JR, et al: Metabolic changes in follicular fluid of the dominant follicle in high- yielding dairy cows early post partum. Theriogenology 2004;62:1131-1143. 25. Perry GA, Swanson OL, Larimore EL, et al: Relationship of follicle size and concentrations of estradiol among cows exhibiting or not exhibiting estrus during a fixed-time AI protocol. Domest Anim Endocrinol 2014;48:15-20. 26. Baratta M,West LA, Turzillo AM, et al: Activin modulates differential effects of estradiol on synthesis and secretion of follicle-stimulating hormone in ovine pituitary cells. Biol Reprod 2001;64:714-719. 27. Miller BG, Moore NW, Murphy L, et al: Early pregnancy in the ewe: effects of oestradiol and progesterone on uterine metabolism and on embryo survival. Aust J Biol Sci 1977;30:279-288. 28. Geisert RD, Zavy MC, Biggers BG, et al: Characterization of the uterine environment during early conceptus expansion in the bovine. Anim Reprod Sci 1998;16:11-25. Clinical Theriogenology • Volume 8 Number 2 • June 2016 108 Figure 1: Schematic presentation of synchronization protocol Figure 1A CIDR Day 0 GnRH PGF2a, Day 7 Day 10 GnRH + TAI 66 h CIDR Day 0 GnRH PGF2a Day 7 Day 10 TAI 48 h GnRH Day 9 18 h Ovsynch + CIDR CO-Synch +CIDR Clinical Theriogenology • Volume 8 Number 2 • June 2016109 Figure 1B Note: In order to inseminate heifers in both LT-72 and ST-72 groups at the same time, the synchronization of heifers in ST-72 group was initiated on Day 25 of LT-72 protocol. Figure 2: Mean (± SEM) artificial insemination (AI) pregnancy percentage in beef cattle inseminated with or without AI gun protective sheath. 59.4 55.9 58.3 56.7 0 20 40 60 80 100 Sheath No sheath Sheath No sheath Cows Heifers (95/160) (90/161) (127/218) (121/212) P > 0.1 PGF2a CIDR PGF2a 6 h FTAI @ 72 h from CDIR removal ST 72 (5-d CO-Synch+CIDR) Day 25 Day 14 Day 30 AM Day 33 GnRH GnRH + FTAI FTAI @ 72 h from CDIR removal PGF2a CIDR Day 0 Day 14 Day 30 Day 33 GnRH + FTAI LT 72 (14-d CIDR – PGF – GnRH) Clinical Theriogenology • Volume 8 Number 2 • June 2016 110 Table 1.Mean ± SEM of age, body condition scores, and days postpartum of beef cows belonging to treatment groups in different locations Cattle groups Variables Treatment n Mean ± SEM Cow Age (yr) CON 161 5.73 ± 0.21 TRT 160 5.58 ± 0.21 BCS CON 161 6.07± 0.08 TRT 160 6.43 ±0.45 DPP CON 161 52.8 ± 0.67 TRT 160 52.9 ± 0.68 Heifers Age (mo) CON 212 15.3 ±0.43 TRT 218 15.6 ± 0.37 BCS CON 212 5.93 ± 0.97 TRT 218 6.12 ± 1.49 RTS CON 212 4.09 ± 1,08 TRT 218 4.14 ± 1.07 SEM, Standard error of the mean; CON, AI gun with no protective sheath; TRT, AI gun with protective sheath; BCS, Body condition score (1 to 9; 1, emaciated; 9, obese); DPP, days postpartum; RTS, Reproductive tract score (1 to 5; 1, immature, anestrus; 5, mature, cycling); Table 2. Effect of explanatory variables on AI pregnancy rates from beef cows and beef heifers that were inseminated with (TRT) and without (CON) the use of a protective plastic sheath (PS) Beef cattle Explanatory variables Degrees of Freedom F value P value Cow Protective sheath 1 0.08 0.78 Synchronization treatment 1 0.41 0.52 Estrus 1 8.07 0.005 Age 2 0.71 0.49 Body condition score 1 0.10 0.79 Days postpartum 1 0.43 0.52 Heifer Protective sheath 1 0.09 0.82 Synchronization treatment 1 0.42 0.52 Estrus 1 9.72 0.002 Age 1 2.86 0.09 Body condition score 1 0.10 0.92 Reproductive tract score 3 0.53 0.66 Refer to Figure 1 for cow and heifer synchronization treatment; Estrus - expression of estrus at or prior to AI vs no expression; Cow age categories (yrs)- 2, 3 to 6, > 6 years; Body condition score (1 to 9; 1, emaciated; 9, obese) categories, ≤ 5 and > 5; DPP, Days postpartum categories ≤ 60 and > 60; Reproductive tract score (1 to 5; 1, immature, anestrus; 5, mature, cycling), categories 2 to 4; Heifer age categories (mo), ≤ 15 and > 15; Cow - Covariance parameter estimates: Location, 0.07831; AI sire, 0.06429; Inseminators, 0.03344; Residual 0.28937; Fit statistics - BIC =1276.06; -2 Res log likelihood =1254.51; Heifer - Covariance parameter estimates: Location, 0.10586; AI sire, 0.08713; Inseminators, 0.08237; Residual, 0.22820; Fit statistics, BIC = 1386.8; -2 Res log likelihood = 1371.8; Clinical Theriogenology • Volume 8 Number 2 • June 2016111 Clinical Theriogenology • Volume 8 Number 2 • June 2016 112 OMNIBLANK: << /ASCII85EncodePages false /AllowTransparency false /AutoPositionEPSFiles true /AutoRotatePages /None /Binding /Left /CalGrayProfile (Dot Gain 20%) /CalRGBProfile (sRGB IEC61966-2.1) /CalCMYKProfile (U.S. Web Coated \050SWOP\051 v2) /sRGBProfile (sRGB IEC61966-2.1) /CannotEmbedFontPolicy /Warning /CompatibilityLevel 1.6 /CompressObjects /Tags /CompressPages false /ConvertImagesToIndexed true /PassThroughJPEGImages true /CreateJobTicket false /DefaultRenderingIntent /Default /DetectBlends true /DetectCurves 0.0000 /ColorConversionStrategy /LeaveColorUnchanged /DoThumbnails false /EmbedAllFonts true /EmbedOpenType true /ParseICCProfilesInComments true /EmbedJobOptions true /DSCReportingLevel 0 /EmitDSCWarnings false /EndPage -1 /ImageMemory 1048576 /LockDistillerParams false /MaxSubsetPct 100 /Optimize true /OPM 1 /ParseDSCComments true /ParseDSCCommentsForDocInfo true /PreserveCopyPage true /PreserveDICMYKValues true /PreserveEPSInfo true /PreserveFlatness false /PreserveHalftoneInfo false /PreserveOPIComments true /PreserveOverprintSettings true /StartPage 1 /SubsetFonts true /TransferFunctionInfo /Apply /UCRandBGInfo /Preserve /UsePrologue false /ColorSettingsFile () /AlwaysEmbed [ true /ArialNarrow /ArialNarrow-Bold /ArialNarrow-BoldItalic /ArialNarrow-Italic ] /NeverEmbed [ true ] /AntiAliasColorImages false /CropColorImages false /ColorImageMinResolution 300 /ColorImageMinResolutionPolicy /OK /DownsampleColorImages false /ColorImageDownsampleType /None /ColorImageResolution 300 /ColorImageDepth -1 /ColorImageMinDownsampleDepth 1 /ColorImageDownsampleThreshold 1.50000 /EncodeColorImages false /ColorImageFilter /DCTEncode /AutoFilterColorImages true /ColorImageAutoFilterStrategy /JPEG /ColorACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /ColorImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000ColorACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000ColorImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasGrayImages false /CropGrayImages false /GrayImageMinResolution 300 /GrayImageMinResolutionPolicy /OK /DownsampleGrayImages false /GrayImageDownsampleType /None /GrayImageResolution 300 /GrayImageDepth -1 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages false /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /GrayImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000GrayACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000GrayImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasMonoImages false /CropMonoImages false /MonoImageMinResolution 1200 /MonoImageMinResolutionPolicy /OK /DownsampleMonoImages false /MonoImageDownsampleType /None /MonoImageResolution 1200 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages false /MonoImageFilter /CCITTFaxEncode /MonoImageDict << /K -1 >> /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile (None) /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False /CreateJDFFile false /Description << /ENU ([Based on '[Press Quality]'] Use these settings to create Adobe PDF documents best suited for high-quality prepress printing. 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