




































 

 

 
87 

† Corresponding author 
© 2015 Conscientia Beam. All Rights Reserved. 

 

IN VITRO DIFFERENTIAL EFFECT OF NERVE GROWTH FACTOR ON 

FUNCTIONAL PARAMETERS OF MURRAH BUFFALO    SPERMATOZOA IN 

LOW AND HIGH FERTILE GROUPS 

 

Namagiri Lakshmi S.1 --- Anand Laxmi N.2† 

 
1Assistant Professor, Veterinary College and Research Institute, Orathnadu, Tamilnadu, India 

2Principal Scientist, Dairy Cattle Physiology division, National Dairy Research Institute, Karnal, Haryana, India 

 

ABSTRACT 

Aim of the present study was to examine the influence of in vitro supplementation of Nerve growth factor 

(NGF) on functional parameters of Murrah buffalo (Bubalusbubalis) spermatozoa from fresh semen, like, 

motility, plasmalemma integrity, acrosomal integrity, ATP concentration. Fresh semen samples (n=6) were 

washed in Tris buffer and divided into two equal parts (control and NGF groups). Only in the NGF 

group, NGF was added to a final concentration of 50 and 100 ng/ml. The samples were incubated at 37o 

C for different time intervals   in TCM 199 medium supplemented with BSA and the effects were observed 

at 0, 30, 60 and 120 min of incubation. The experiment was performed in low (LF) and high fertile(HF) 

groups based on previous three years conception rate and taking cutoff value , below and above this value  

were categorized as  LF and HF group. The mean concentration of the buffalo seminal plasma (n=12) 

NGF was 67.7±3.25ng/ml and 65.5± 2.76 in LF and HF groups respectively. The concentration of 

NGF in blood plasma was 83.5±7.82 and 68.6±3.82 in HF and LF groups respectively. The 

concentration of blood plasma NGF being higher (P<0.05) in HF group. With either dose of NGF in 

vitro significant effect on the total motility (P<0.05), progressive forward motility (P<0.05) was observed. 

It could be maintained in HF group till 120 min but in LF group it was restricted to 60 min when 

compared with their respective control. The functional membrane integrity did not differ significantly 

between groups (control and NGF treated) in both LF and HF groups with either concentration of NGF. 

The plasma lemma integrity was significantly less (P<0.05) at 120 min of incubation when compared with 

the initial value at 0 min of incubation. The percentage of acrosomal intact spermatozoa decreased 

continuously over a period of time in both the groups. As compared to 0 min of incubation, the significant 

(P<0.05) loss of acrosome was observed at 60 and 120 min of incubation in LF and HF control groups 

Animal Review 
2015 Vol. 2, No. 4, pp. 87-98 
ISSN(e): 2409-6490 
ISSN(p): 2412-3382 
DOI: 10.18488/journal.ar/2015.2.4/101.4.87.98 
© 2015 Conscientia Beam. All Rights Reserved. 

 
 
 
 

http://crossmark.crossref.org/dialog/?doi=10.18488/journal.ar/2015.2.4/101.4.87.98


Animal Review, 2015, 2(4): 87-98 

 

 
88 

© 2015 Conscientia Beam. All Rights Reserved. 

and NGF supplementation could maintain acrosome integrity in HF group for 60 min where as in LF 

group it could be maintained significantly only till 30 min of incubation when compared with the initial 

values of respective groups. Viability of spermatozoa was not significantly different when compared between 

groups with their respective control, However  when compared at different time intervals, the viability of 

sperms in HF and LF supplemented  groups was significantly different from initial values only at 120 min 

of incubation whereas in respective control groups the loss in viability was significant from 60 min itself. 

With respect to ATP concentration of spermatozoa in different groups it was observed that 100ng dose 

could increase the concentration of ATP in HF group significantly (P<0.05) at 60 min of incubation only 

with NGF in vitro when compared with its respective control. In conclusion, all the parameters decreased 

significantly at 120 min of incubation when compared with their respective initial values for all the groups. 

In HF group supplementation of NGF@50ng/ml could maintain functional parameters of spermatozoa 

for a greater duration of time when compared with LF or control group.  

Keywords: Spermatozoa, Nerve growth factor, Murrah buffalo, Fertility, Time of incubation. 

 

Received: 30 August 2015/ Revised: 30 September 2015/ Accepted: 5 October 2015/ Published: 10 October 2015 

 

1. INTRODUCTION 

Although various neurotrophins have been detected in mammalian testis [1] only NGF 

seems to have a potential role in male reproduction [2]. Report of Server and Shooter [3] 

demonstrated the source of Nerve growth factor (NGF) as sub mandibular gland of adult mouse 

but later presence and purification of NGF was done from seminal plasma of bovine species and 

was partially characterized.  

The biological unit of NGF was observed to be more per ml of semen in bovine, when 

compared with the activity of NGF of other ruminants and human [4]. In buffaloes no work has 

been carried out, hence the present study was undertaken for estimating the concentration of 

blood and seminal plasma NGF in low and high fertile group and study in vitro effect of NGF on 

buffalo spermatozoa functional parameters. Studies on NGF/TRKA system indicated it’s role in 

the physiology of male reproduction [5-7]. Exogenous NGF as cry oprotectant improved sperm 

viability and motility, increased intracellular NO concentration, and decreased apoptosis content 

in normal human spermatozoa [8] NGF, belongs to a family of neurotrophins, whose role is in 

maintaining development of neurons. Their role is not only restricted to nervous system, but they 

also have role in regulating reproductive system.  

It has been well studied in humans [9-11]. Extensive work by Li, et al. [12] 

immunolocalized the receptor on ejaculated bovine spermatozoa. The study also reported that 

exogenous supplementation of NGF to bovine sperm samples increased leptin secretion, sperm 

viability when concentration of NGF was increased from 20-120 ug/L. In humans, it’s differential 

level has been reported and lower level has been related with respect to inferior quality of the 

semen [2]. In the present study, a comprehensive study has been done to estimate the blood and 

semen plasma level of NGF, localize, NGF receptors in the spermatozoa of high and low fertile 



Animal Review, 2015, 2(4): 87-98 

 

 
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Murrah buffalo bull. In vitro effect of NGF @ 50ng and 100ng on spermatozoa functional 

parameters was also evaluated. 

 

2. MATERIAL AND METHODS 

Enzyme immunoassay kit (EIA) for estimation of NGF in blood and seminal plasma was 

purchased from WKEA MED. supplies Corporation, China. For estimation of ATP in 

spermatozoa samples, kit was purchased from Abnova Co., Germany. The primary antibody used 

against NGF receptor was purchased  from Biorbyt Ltd., UK. The FITC conjugated secondary 

antibody was purchased from Chromus Co., Bengaluru, INDIA. For analyzing the PCR products 

for the NGF receptor and for capturing the image of gel with products , Alpha Digidoc, 

documentation system was used.  

 

2.1. Grouping of Murrah Buffalo Bulls  

A total of 15 Murrah buffalo bulls from Artificial Breeding Research Center, NDRI were 

selected for the present study. They were maintained under routine management conditions and 

were fed ad libitum. Data for total number of services for each bull was collected from the records 

available at the center. Number of services, for these bulls ranged from 46 to 85. Baseline for 

number of services was considered as 46. The conception rate was calculated based on first 46 no. 

of services of each bull. The conception rate obtained with semen of the selected bulls was fitted 

in normal distribution, and they were divided into high, medium and low fertile groups. Average 

CR was 36.57% and SD value was 7.54. High and low fertile bulls were selected with higher and 

lower CR than the estimated value. Six bulls for each high (HF) and low fertile (LF) groups were 

selected. Mass activity of the semen sample was also considered, average being 2.7 ± 0.52. After 

grouping, average values for different parameters like body weight (Kg), concentration of sperms 

(millions/ml) , testosterone (ng/ml) and other factors were also estimated/recorded for both the 

groups (Table A). 

 

Table-A. Comparison of different parameters between two groups of Murrah bulls 

Parameters Low fertile High fertile 

Body weight (Kg) 685±9.0 704±7.2 
Concentration of sperms (106/ml) 849±12 875±32 
Concentration of testosterone  (ng/ml)              1.85±0.14 2.09±0.17 
Conception rate(%) 28±4.05 44±2.86 

Average volume of semen (ml) 2.9±0.54 3.2±0.28 

 

2.2. Collection of Blood 

For estimation of testosterone and nerve growth factor (NGF) in blood and seminal plasma 

samples, blood was collected by jugular vein puncture once in 15d for two months. Immediately 

after collection, blood samples were centrifuged at 3000 rpm, plasma was separated and stored at 

-20oC until assay was done. 

 



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2.3. Collection of Semen Samples and Processing  

For assay of NGF in semen samples, fresh semen sample was collected from Murrah bulls six 

number, each in HF and LF group using sterile artificial vagina (CMV France). Hygienic 

conditions were followed during collection of semen. After collection of semen, samples were 

centrifuged at 5585 g for 10 min at 4oC to separate out the supernatant fraction containing 

seminal plasma. The seminal plasma samples were also stored at -20oC.Spermatozoa fraction was 

also separated out, the fraction was washed with Tris buffer (pH 7.2 ). Then the sperm samples 

were suspended in TCM 199 medium to a concentration adjusted to 10 million sperms/ml. 

 

2.4. Evaluation of Different Spermatozoa Functions 

Sperm motility was estimated at 37°C, by examination of a wet mount using bright-field 

microscopy (400X). Sperm viability was determined by preparing an eosin-nigrosin smear (37°C) 

and assessing at least 100 sperm under bright-field microscopy at 1000X; [13].  

Acrosome reaction evaluations were carried out using 10μL-semen smears that had been 

stained with Trypan-Blue/Giemsa (TB) according to Kitiyanant, et al. [14]. In each smear at 

200x magnification, 200 spermatozoa were analyzed and two distinct spermatozoal classes were 

identified: 1) intact 2) damaged. 

Sperm plasma membrane integrity was evaluated using supravitalhypoosmotic swelling test. 

After the incubation of HOS, equal drop of HOS solution and eosin [0.5% (w/v), sodium citrate 

2.92%] was placed on a warm slide, mixed for 10 seconds and cover slip was placed before the 

evaluation for plasma membrane integrity under phase contrast microscope at 400X. A total of 

one hundred spermatozoa were observed in at least five different fields. Clear heads and tails and 

swollen tails were considered intact with biochemically active sperm membranes, while pink 

heads and tails and unswollen tails were considered disrupted, inactive sperm membranes. Hypo-

osmotic swelling (HOS) assay was performed as described by Ramu and Jeyendran [15]. 

 

2.5. IN VITRO Studies 

2.5.1. Supplementation of NGF for in Vitro Studies 

After separating out seminal plasma from ejaculated semen, the precipitate fraction 

containing spermatozoa was dispersed in TCM 199 medium and they were adjusted to ten million 

number of sperms in one ml of the medium. Nerve growth factor was supplemented @ 50 ng and 

100 ng/ml for three different time periods of incubation viz. 30, 60 and 120 min for both LF and 

HF groups. Percentage of motile or viable spermatozoa were estimated and spermatozoa were 

also evaluated for acrosome integrity, plasmalemma integrity by HOST test and ATP production. 

Each trial was conducted in triplicate and four trials for each parameter was conducted.  

 

2.5.2. Localization of NGF Receptor by Immunofluorescence  

For localizing receptor, spermatozoa were fixed with 100% methanol for 30 min followed by 

washing with PBS. Subsequently sperms were incubated with mouse monoclonal antibody specific 



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91 

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for human NGF receptor (1: 100 dilution) or with PBS (for control) for 3 h followed by washing 

with PBS. Specimens were then incubated with FITC-conjugated secondary antibody (anti-rabbit 

IgG FITC conjugate, 1:100 dilution) for 90 min. at 37oC. Further, samples were treated with 1,4-

diazabicycol (2.2.2) octane, covered with a cover slip and sealed. Specimen were examined under a 

fluorescence microscope at 40 X magnification and  as described by Li, et al. [2]. 

 

2.5.3. Confirmation of Receptor by PCR 

Polymerase chain reaction was also performed for localizing the receptor. DNA was 

extracted from spermatozoa samples using modified phenol chloroform extraction method [16]. 

A 100 ng DNA was precipitated out from 40-50 X106 spermatozoa. A100 ng of extracted DNA 

was amplified with the NGF primers (Table-B) by PCR with different componants of PCR 

mixture as given in (Table-C)as per the time for different events as given under reaction 

programme for PCR. PCR product obtained was evaluated by electrophoresis on a 1.8% agarose 

gel [17]. Purity of the DNA was estimated by taking the ratio of the absorbances recorded at 260 

nm and 280 nm. The amount of DNA (ng/ml) present was calculated as O.D. 1 at 260 nm is 

equivalent to 50 ng ds DNA/ul). 

 

Table-B. Sequences of forward and reverse primers 

Receptor Primer Selection  Fragment size 

NGF r 
(TrKA) 

5’-CTGGGTGAGGGTGCCTTT 
3’-CGCTCAGACACCTCCTTCAG 

112 bp 

 

Table-C. Concentration of different componants in  PCR mixture   

Contents Quantity 

Total mixture 50ul 
Genomic DNA   2ul 
Primer (F) 0.5ul 
Primer  (R) 0.5ul 
Master mix 25ul 
MilliQ water 22ul 

 

Reaction Programme for PCR 

Initial denaturation   95oC  2 min. 

Denaturation    95oC  30 s 

Annealing    59oC                  30s 

Extension    72oC  45 s. 

 

The step was continued for 35 cycles and final extension was carried out at 72oC for 4 min. 

PCR was carried out in thermocycler (QB96, Quanta Biotech., UK). 

 

 

 



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2.6. Statistical Analyses 

All analyses were done using software package. Data from different experiments are 

presented a Mean±SEM. Significance of the parameters was evaluated by using three way 

ANOVA considering concentration of supplemented NGF in vitro ,Incubation time and group 

factors. 

 

3. RESULTS 

Blood plasma concentration of NGF was observed to be higher (P<0.05) in HF group when 

compared with LF group. The concentration of NGF in seminal plasma when compared between 

groups, the difference was not significant. The Mean±SEM concentration of testosterone was 

also not significantly different between the groups (Table 1).The intra and interassay CV 

percentage as analyzed for hormone estimations was always <10%. 

 
Table-1. Concentration of NGF in blood and seminal plasma and testosterone in blood plasma of low   and high fertile 
groups 

 

Values are expressed as Mean±SE. Values with capital superscripts differ significantly (P<0.05) within a row. 

 

3.1. In Vitro Supplementation of NGF on Percentage of Motile Sperms 

When 10 million spermatozoa were treated with different concentrations of 50 or 100 ng 

NGF, change in the percentage of motile spermatozoa was not significant in LF or HF group 

when compared with their respective control, when time factor was kept constant. Although at 60 

min, it was observed that supplementation with either dose could maintain significantly greater 

percentage of motile sperms. When the motility parameter was estimated for different time 

intervals of incubation, with either  dose of NGF, it was observed that there was decrease in the 

percentage of motile sperms with increase in the time of incubation but the decrease was 

significant (P<0.05) only at 120 min of incubation in HF group, where as in LF control group it 

was significant as early as at 60 min and was further significant also at 120 min (P<0.05), but 

such further significant decrease could not be observed at 120 min, in HF control group  samples 

(Table 2) . 

 
Table-2. Effect of in vitro supplementation of NGF (ng/ml) on percentage of motile sperms 

 
Values are expressed as Mean±SE. Values with small superscripts differ significantly (P<0.05) within a column. 

 

 



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3.2. In Vitro Supplementation of NGF on Percentage of Viable Spermatozoa 

On addition of NGF to HF/LF group spermatozoa samples; there was no significant effect on 

percentage of viable spermatozoa when time factor was kept constant except at 60 min 

supplementation increased the viability in HF group when compared with the viability of sperms 

of respective control group. When concentration of NGF @ 50 or 100ng was kept constant, it 

was observed that in both LF and HF groups percentage of viable sperms decreased significantly 

(P<0.05) only at 120 min of incubation, but without supplementation in the control group, 

viability could be maintained only till 30 min without significant change but further from 60 min 

the decrease was significant (P<0.05) from initial value (Table 3). 

 
Table-3. Effect of in vitro supplementation of NGF (ng/ml) on percentage of viable sperms 

 
Values are expressed as Mean±SE. Values with capital superscripts differ significantly (P<0.05) within a   row. Values with small 
superscripts differ significantly (P<0.05) within  a column. 

 

3.3. In Vitro Supplementation of NGF on Acrosome Integrity 

When time factor was kept constant, within a group the acrosome integrity of spermatozoa of 

supplemented groups was not significantly different from the acrosome integrity of control group. 

On supplementation of either dose of NGF to spermatozoa of LF/HF group, the decrease in 

acrosome integrity observed was not significant, till 60 min of incubation from the initial value, 

but decreased significantly (P<0.05) at 120 min. In the LF control group samples, the values were 

significantly less (P<0.05) at 60 min of incubation and could be maintained only till 30 min. 

Whereas in HF control group acrosome integrity of spermatozoa could be maintained till 60min 

but at 120 min it decreased significantly (P<0.05)(Table 4). 

 

Table-4. Effect of in vitro supplementation of NGF (ng/ml) on percentage of  sperms with acrosome integrity 

 
Values are expressed as Mean±SE. Values with small superscripts differ significantly (P<0.05) within a column.  

 

3.4. In Vitro Supplementation of NGF on Plasmalemma Integrity 

 In control group without supplementation of NGF, the integrity of plasmalemma as assessed 

by HOST test decreased with increase in the time of incubation, the decrease was significant 



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(P<0.05) at 120 min of incubation. Similar was the trend observed in LF group on 

supplementation with either 50 or 100 ng dose of NGF.  

Keeping the time factor constant, the plasmalemma  integrity of spermatozoa was not 

significantly different from control when any time period of incubation was considered. In HF 

group supplementation of NGF did not alter the integrity of plasmalemma of spermatozoa 

significantly at different fixed time intervals when compared with the respective  control. Similar 

were the results when compared with the values at initial time period of incubation and also  at 

different time intervals keeping dose factor constant (Table 5). 

 

Table-5. Effect of in vitro supplementation of NGF (ng/ml) on percentage of sperms with plasmalemma integrity 

   Values are expressed as Mean±SE. Values with small superscripts differ significantly (P<0.05) within a column. 

 

2.5. In Vitro Supplementation of NGF on Concentration of Spermatozoa ATP 

The concentration of ATP in spermatozoa decreased significantly, as the time of incubation 

increased irrespective of whether the group was supplemented or not supplemented. When the 

time factor was kept constant, only at 60 min of incubation, supplementation with 100 ng dose of 

NGF in HF group could increase the concentration of spermatozoa ATP, which was 

significantly(P<0.05) different from control, where as supplementation could not increase the 

concentration  of ATP significantly at 30 or 120 min of incubation (Table 6). 

 

Table-6. Effect of in vitro supplementation of NGF (ng/ml) on concentration of ATP in spermatozoa lysates 

Values are expressed as Mean±SE. Lysate of 10x106 sperms was used. Values with different capital superscripts differ significantly (P<0.05) 

within a row. 

 

When all the parameters were compared between low and high fertile groups, keeping time 

factor constant at a time , it was observed that all the parameters were greater for HF group 

when compared with LF group, although were not significantly different, except at 60 min for 

motility, viability and concentration of spermatozoa ATP. When different parameters were 

Time of  
Incubation 

                              LF                                 HF      

Dose of NGF supplemented Dose of NGF supplemented 

0 50 100 0 50 100 

0 58.08a±1.02 58.45a±1.00 58.54a±1.03 65.25±1.02 65.86±0.99 68.83±0.95 
30 56.23±0.99 56.89±1.02 56.89±0.98 64.23±1.01 65.23±1.00 67.45±0.98 
60 56.45±0.96 56.23±0.99 56.45±0.92 63.32±0.98 64.89±0.97 67.23±0.96 
120 46.98b±0.85 50.23b±0.75 49.87b±1.02 60.52±1.03 64.42±1.02 65.01±0.88 

Time of  
Incubation 

                              LF                                 HF      

Dose of NGF supplemented Dose of NGF supplemented 

0 50 100 0 50 100 

0 6.417±0.02 6.421±0.02 6.454±0.03 6.425±0.03 6.423±0.02 6.419±0.02 
30 6.361±0.02 6.328±0.02 6.388±0.02 6.342±0.02 6.352±0.03 6.360±0.03 
60 6.309±0.01 6.305±0.02 6.308±0.03 6.325A±0.03 6.320±0.02 6.358B±0.02 
120 6.208±0.02 6.222±0.01 6.231±0.02 6.210±0.02 6.221±0.02 6.225±0.01 



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compared for different time periods of incubation, in both high and low fertile group, all the 

parameters decreased significantly with increase in the time period of incubation, except for one 

parameter namely plasmalemma integrity which was not significantly different at any time period 

of incubation .In HF group supplementation of spermatozoa with NGF could maintain motility, 

viability and acrosome integrity for greater period of time i.e. 60 min whereas in LF 

supplemented groups it could be maintained till 30 min only . 

By immunoflourescence technique, using fluorescent antibody, it was observed that 

fluorescence was intense at the acrosomal and post acrosomal regions, irrespective of high or low 

fertile group. Fluorescence could be observed in both the groups (Fig 1). The test was qualitative. 

When the extracted spermatozoa DNA was subjected to PCR, and run on 1.8 % agarose gel a 112 

bp product was obtained for both LF and HF groups confirming the presence of receptor (Fig 2). 

 

 

Fig-1. Immunolocalization of NGF receptor in Murrah 
buffalo bull spermatozoa 

Fig-2. PCR product of NGF receptor (112 bp) 
L-  50 bp ladder LF (1-    8),HF   (9-12)   
P- positive marker for NGF receptor 

 

3. DISCUSSION 

There is evidence that a neurotropin like NGF has important role in non neuronal cells in 

reproductive system [18]. In the present study, higher concentration of blood plasma NGF of HF 

group indicates positive relation between plasma concentration of NGF and spermatozoa 

functional parameters and fertility of bulls. In the sperm lysate, NGF could not be detected by 

EIA. Reports of Li, et al. [19]; Li, et al. [2] have suggested that NGF is present in spermatozoa, 

but the methodology for estimation was by Western blotting and PCR. In the present study it 

was observed that all the parameters related to spermatozoa, were higher in the HF when 

compared with LF group. At the same time, concentration of blood plasma NGF was higher in 

HF group. It suggests regulatory role of circulatory NGF on buffalo sperm functional 

parameters. The physiological concentration of NGF (50/100 ng/ml) was more closely related in 

augmenting sperm physiological functions in vitro in the present study. Li, et al. [2] reported the 

same in bovines and humans. With immunofluorescence technique it was observed that TrKA 

receptor is present  on spermatozoa and PCR studies confirmed the presence of NGF receptor on 

Murrah bull spermatozoa. Using an immunofluorescent technique, NGF-immunoreactivity was 

localized to the sperm head and tail, whereas that of TrkA was detected in the acrosomal cap, 



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nucleus, and tail regions [2, 20].This is the first report in Murrah bull confirming presence of 

NGF receptor in spermatozoa. In vitro studies regarding effect of NGF on human sperm motility 

by CASA showed that sperm motility were enhanced in a time and dose dependant manner [21]. 

Study by Saeednia, et al. [8] reported that addition of NGF to medium containing post thawed 

sperms can enhance motility and viability of human sperms. Hence from the present study, it can 

be suggested that supplementation of NGF in vitro might have enhanced buffalo spermatozoa 

viability and motility in both LF and HF groups till one hour of incubation when compared with 

the non supplemented group. This study was carried out for short duration of time; technology 

has to be improvised for conducting experiments for longer durations. Report of Perrard, et al. 

[22] suggested that NGF is produced by germ cells and acts on sertoti cells. In the present 

study, however, by EIA technique, we could not measure NGF in sperm lysates. May be other 

techniques can demonstrate the presence/secretion of NGF by Murrah bull spermatozoa. It’s 

known that TRKA binds NGF and its presence could be detected in buffalo bull spermatozoa, 

suggesting role of circulatory/seminal plasma NGF in regulating spermatozoa function. This was 

confirmed by in vitro studies with supplementation of NGF on different sperm functions. With 

respect to spermatozoa integrity and viability on NGF supplementation in vitro, it was observed 

that NGF could maintain viability/reduced permeability of membrane and plasmalemma integrity 

of spermatozoa for a greater duration of time in vitro when compared with control within 120 min 

of study. Although the extent of  its effect in augmenting functions or reducing the damage to the 

membrane was different when compared between  LF and HF group . Hence its effect was more 

prominent in maintaining spermatozoa functional parameters of HF group when compared with 

the functional parameters of LF group. The mechanism by which NGF affects the viability and 

motility parameters is not understood and study is required in this area. This study also suggests 

that enhancement of the sperm functions by supplementation of NGF in vitro may be applicable in 

assisted reproductive technologies yielding better results with Murrah bull spermatozoa. 

 

Funding: This study received no specific financial support. 
 

Competing Interests: The authors declare that they have no competing interests. 
 

Contributors/Acknowledgement: Both authors contributed equally to the conception and design of the 
study.  

 

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