Comparative RNA isolation methods from fresh ejaculated spermatozoa in Sahiwal cattle (Bos indicus) and Murrah buffalo (Bubalus bubalis) bulls for high quality and enhanced RNA yield.

Sperm mRNA transcriptional profiling can be used to evaluate the fertility of breeding bulls. The aim of the study was to compare the modified RNA isolation methods for higher RNA yield and quality from freshly ejaculated sperm of cattle and buffalo bulls. Ten fresh ejaculates from each Sahiwal (n = 10 bulls × 10 ejaculates) and Murrah bulls (n = 10 bulls x 10 ejaculates) were used for RNA isolation. From the recovered live sperm, total sperm RNA was isolated by conventional methods (TRIzol, Double TRIzol), membrane-based methods combined with TRIzol (RNeasy + TRIzol) with the addition of ß-mercaptoethanol (BME) and Kit (RNeasy mini) methods in fresh semen. Among different isolation methods; the membrane-based modified methods combined with TRIzol (RNeasy + TRIzol) with the addition of ß-mercaptoethanol (BME) resulted significantly (p < .05) higher total RNA quantity (300-340 ng/µL) and better purity in different concentrations of spermatozoa viz., 30-40 million, 70-80 million and 300-400 million sperm. The study concluded that the inclusion of BME to the combined membrane-based methods with somatic cell lysis buffer solution was best for constant increased yield and purity of RNA isolation from Sahiwal cattle and Murrah buffalo bull sperm.

Filtration techniques are advantageous over colloidal centrifugation in improving freezability of low-quality buffalo bull (Bubalus bubalis) ejaculates.

The study compared efficacy of three sperm selection techniques in improving freezability of low-quality Murrah buffalo bull ejaculates. Sephadex (SEP), Sephadex ion-exchange filtration (SIE), and 40/80% BoviPure™ (BP) gradient centrifugation protocols were standardized (ejaculates, n = 24). In Experiment-I, Sephadex G-75, G-100, and combined Sephadex G (75-100) column filtrates were compared. In Experiment-II, BP protocols: 200 g-10 min, 250 g-5, and 10 min, 300 g-10, and 15 min were compared. In fresh semen, Sephadex G (75-100) filtration and 250 g-5 min BP protocol improved sperm functions and were used in Experiment-III, where SEP G (75-100), SIE G (75-100), and 250 g-5 min BP processed ejaculates (n = 48) were cryopreserved and compared at post-thaw stage. The mean recovery rate differed in order: SEP > SIE > BP. SIE filtration significantly improved progressive motility, livability, membrane integrity, bovine cervical mucus penetration and live non-apoptotic sperm. Compared with control, all three techniques equally reduced post-dilution and post-thaw lipid peroxidation (LPO) rate. SEP post-thaw filtrates observed lower cryocapacitation-like changes, LPO (C11-BODIPY581/591), and higher active mitochondria than other treatments. SIE and SEP equally improved post-thaw acrosome-intact sperm over BP. Filtration techniques, preferably, Sephadex ion-exchange filtration can most efficiently process low-quality buffalo bull ejaculates for cryopreservation and improve freezability.

Seasonal and climatic factors have a significant influence on fertility associated sperm phenomic attributes in crossbred breeding bulls (Bos taurus × Bos indicus).

Although seasonal variations in semen quality and fertility have been studied to a considerable extent in breeding bulls, the effect of climatic variables on sperm functional competency has not been understood in detail. The present study analyzed sperm functional parameters in breeding bulls, over a period of 1 year, and assessed the effect of climatic variables on fertility associated sperm parameters. Seasons were categorized into summer, rainy, autumn, and winter based on the meteorological data. Semen was collected from crossbred bulls (n = 7) across the seasons and evaluated for functional membrane integrity, acrosome reaction status, protamine deficiency, capacitation, and lipid peroxidation status using specific fluorescent probes. The results of the present study revealed that bulls produced higher (p < 0.05) viable and acrosome intact spermatozoa during the autumn. The proportion of uncapacitated spermatozoa was also higher (p < 0.05) during autumn. Further, correlation of sperm functional attributes with environmental variables revealed that sperm viability was significantly (p < 0.05) and negatively correlated with daylength and temperature; acrosomal integrity was significantly (p < 0.05) and negatively correlated with day length; and protamine deficiency had significant (p < 0.05) positive correlation with day length and average temperature, and negative correlation with relative humidity. It was concluded that semen produced during autumn was superior to the semen produced during other seasons in terms of sperm functional competencies required for fertility.

Cholesterol-loaded cyclodextrin attenuates dilution effect and improves quality of bovine low sperm insemination doses during cryopreservation.

In the present study, the effect of cholesterol-loaded cyclodextrin (CLC) on the quality of low sperm doses at post-thaw was evaluated. Twenty four ejaculates (6 from each bull) were collected and split into eight aliquots. First four aliquots were diluted up to 20-, 15-, 10- and 5-million sperm/0.25 ml, and remaining four were treated with CLC at the rate of 1 mg/120 million spermatozoa, followed by dilution up to 20-, 15-, 10- and 5-million sperm/0.25 ml. The diluted semen was equilibrated, cryopreserved and evaluated post-thaw. The averages of total motility, progressive motility, average path velocity, straight linear velocity, membrane intact spermatozoa and noncapacitated spermatozoa were higher (p < .05) in CLC-treated sperm doses compared to control ones. However, the moribund spermatozoa, capacitated spermatozoa and acrosome-reacted spermatozoa were reduced (p < .05) in CLC-treated spermatozoa compared to control. The curvilinear velocity and linearity did not differ (p > .05) between control and CLC-treated sperm doses. In conclusion, treatment of spermatozoa with CLC at the rate of 1 mg/120 million spermatozoon attenuates the dilution effect and improves the quality of bovine low sperm insemination doses during cryopreservation; hence it could be a favourable cryoprotectant for preserving bovine semen at higher dilutions.

Spermatozoa produced during winter are superior in terms of phenotypic characteristics and oviduct explants binding ability in the water buffalo (Bubalus bubalis).

Although reduced reproductive efficiency during summer has been well documented in buffaloes, the reason for the same is yet to be understood. The present study was conducted to identify the subtle differences in sperm phenotypic characteristics (motility, membrane integrity, acrosome reaction and lipid peroxidation status), oviduct binding ability and expression of fertility-associated genes (AK 1, ATP5D, CatSper 1, Cytochrome P450 aromatase, SPP1 and PEBP1) between winter and summer seasons in buffaloes. Cryopreserved spermatozoa from 6 Murrah buffalo bulls (3 ejaculates/bull/season) were utilized for the study. Real-time quantitative PCR was performed for assessing the expression patterns of select fertility-associated genes. The proportion of motile and membrane intact spermatozoa was significantly higher (p < .05) in winter as compared to summer ejaculates. The proportion of moribund and lipid peroxidized spermatozoa was significantly lower (p < .05) in winter ejaculates as compared to summer. The sperm-oviduct binding index was significantly lower (p < .01) when spermatozoa from summer ejaculates were used as compared to winter ejaculates. The expression of fertility-associated genes did not differ significantly between the two seasons except for PEPB1; the transcriptional abundance of PEPB1 was significantly (p < .05) lower in summer as compared to winter season. It was inferred that buffalo spermatozoa produced during winter season were superior in terms of cryotolerance, membrane and acrosome integrity, lipid peroxidation status and the ability to bind with oviduct explants.

Sperm functional attributes and oviduct explant binding capacity differs between bulls with different fertility ratings in the water buffalo (Bubalus bubalis).

We report here the differences in sperm functional attributes and sperm-oviduct binding index in bulls with different field fertility ratings. Cryopreserved spermatozoa from Murrah buffalo bulls (n=9) with different fertility ratings were evaluated for membrane integrity, capacitation status, acrosome intactness and protein tyrosine phosphorylation status. Frozen--thawed spermatozoa were incubated with oviduct explants for 1h under 5% CO2, 38.5°C with 95% relative humidity and the number of spermatozoa bound to the unit area of oviduct explants (binding index; BI) was assessed using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) fluorescent staining. The proportion of membrane-intact and acrosome-intact spermatozoa was significantly (P<0.05) higher and the proportion of capacitated spermatozoa was significantly (P<0.05) lower in high-fertile bulls compared with medium- and low-fertile bulls. The relationship between BI and bull fertility was significant and positive (r=0.69; P=0.04). BI was negatively and significantly (r=-0.83; P=0.01) related to membrane-compromised spermatozoa. It was concluded that the sperm-oviduct explant binding index was positively related to (1) the proportion of membrane-intact spermatozoa in a given semen sample and (2) invivo fertility of the buffalo bull, indicating the possibility of developing a fertility prediction tool using a sperm-oviduct explant binding model, once validated on a greater number of bulls.

Sperm protein carbonylation.

The cryopreservation of sperm is a well established technique that plays an essential role in dissemination of elite germplasm of livestock. Despite having numerous advantages, the cryopreservation induces certain stresses on sperm including structural and functional damages leading to impaired sperm quality and fertility, which might be associated with production of reactive oxygen species (ROS). In addition, the ROS upon reacting with sperm lipids, DNA and proteins may lead to a cascade of sperm damages. The sperm membrane contains a rich amount of polyunsaturated fatty acids, which increases their susceptibility to oxidative stress induced damages, leading to formation of secondary products. These secondary products result in oxidation of sperm proteins via carbonylation. The carbonylation could lead to disturbances in specific proteins that are involved in capacitation. The present review deals with sperm protein carbonylation.

Effect of automatic cluster remover settings on milkability, milk quality and milking irregularities of crossbred cows.

Automatic cluster remover (ACR) settings regulate the end of milking by detaching the clusters based on milk flow dropping below a preset level, which needs to be standardised for different breeds of dairy animals based on their production. A study was conducted to find out the best ACR setting for milking Indian crossbred cows based on milkability, milking irregularities and milk quality. Fifty six crossbred dairy cows in lactations 1 to 4 were categorised into three groups based on the level of production; low (N = 16; 18 kg/d). The ACR settings tested were 0.1, 0.2, 0.3 and 0.4 kg/min, keeping the vacuum level and pulsation settings constant. The ACR settings significantly (P < 0.01) affected the milk yield at all levels of production with a significant effect (P < 0.01) on machine-on time at 0.4 kg/min. The yield during the first 2 min of milking, average flow and peak flow rates were not affected at any level of production. The average electrical conductivity in milk was significantly (P < 0.01) lower for the low and medium yield cows without affecting the mean somatic cell count. At 0.4 kg/min, more cluster reattachments were needed because of significant amount of milk remaining in the udders post-cluster removal.

Effect of Tris-egg Yolk, Soya Milk, and Liposome-based Extenders on Sahiwal (Bos indicus) Sperm Quality during Pre- and Post-Cryopreservation Stages.

BACKGROUND: Even though there are plenty of semen cryopreservation extenders available, their adoption is limited. Although normal tris-based egg yolk (EYC) extender is widely used, it leads to compromised post-thaw sperm quality. OBJECTIVE: To find a standard semen extender, six different semen extenders were validated. METHODS: In a split study, six aliquots of zebu cattle fresh semen ejaculate were cryopreserved in extenders containing egg yolk obtained from hen which was reared either in 1) normal, 2) omega-3 enriched, and 3) herbal enriched diet supplementation, and egg yolk free extenders such as 4) soya lecithin, 5) Bioxcell and 6) Optixcell. RESULT: Significantly poor sperm quality and kinematics were observed in extender containing herbal egg yolk. However, omega-3 enriched egg yolk extender was on par with EYC. Among all extenders, soya lecithin and bioxcell have shown better sperm quality. Sperm motility was significantly higher in semen extended in liposome-based extender Optixcell. CONCULSION: Optixcell can be considered as a standard extender for cattle semen cryopreservation to maintain adequate sperm quality required for artificial insemination.

Influence of season and climatic variables on testicular cytology, semen quality and melatonin concentrations in crossbred bucks reared under subtropical climate.

Seasonality in reproduction and effects of climatic variables on testicular cytology and semen quality in bucks reared under subtropical climatic conditions were not well understood. In the present study, using testicular cytology, semen evaluation and melatonin concentrations assessed over a period of 1 year, we report that bucks reared under subtropical climatic conditions did not show seasonality in reproduction. Climatic variables including temperature, relative humidity, temperature-humidity index (THI), sunshine hours and day length were recorded daily during the whole period of experimentation (one complete year). Ejaculates were collected from crossbred (Alpine X Beetal) males (n = 6) biweekly using artificial vagina, and semen quality (volume, mass activity, sperm concentration, motility, viability, membrane integrity and protamine deficiency) was assessed. To understand the seasonal influence at testicular level, using fine needle aspiration biopsy method, testicular cells were aspirated and different types of cells and testicular cytology indices were quantified. Blood was collected biweekly for estimation of melatonin concentrations. Mass activity was higher (P < 0.05) during rainy season while individual sperm motility and sperm concentration were higher (P < 0.05) during rainy and autumn seasons as compared to other seasons. Sperm functional parameters did not show any differences during different seasons. Sertoli cell count, spermatogenic cell count and testicular indices did not differ among the seasons. Melatonin concentrations also did not differ significantly among the four seasons studied. Among the climatic parameters, THI had significant (P < 0.05) influence on sperm quality. The proportion of Sertoli cell in the testicular cytology had a significant and positive relationship with RH, THI and day length. It was concluded that seasonal variations are less evident in terms of spermatogenesis and semen quality in Alpine X Beetal crossbred bucks reared under subtropical climatic conditions.

Functional characterization of Mammary Gland Protein-40, a chitinase-like glycoprotein expressed during mammary gland apoptosis.

MGP-40 is a chitinase-like protein which is over expressed during mammary gland involution. However, its physiological function in the mammary gland is poorly understood. In the present investigation, we have reported the functional significance of buffalo specific MGP-40 in the mammary gland by using an in vitro model of the buffalo mammary epithelial cell (BuMEC) line. MGP-40 was highly up regulated in BuMECs in serum starved condition as well as after treatment with prolactin suggesting its role in the stress response. Subsequently, to study the effect of MGP-40 on BuMECs, the cells were transfected with a mammalian expression construct of pCI neo harboring MGP-40 gene. It was observed that over expression of MGP-40 enhanced proliferation of BuMECs and protected the cells from apoptosis under serum free condition. In contrast, MGP-40 attenuated the mitogenic effect of insulin in BuMECs. Besides, over expression of the MGP-40 reduced dome formation, acinar polarization and casein synthesis in BuMECs in the presence of lactogenic hormones, it also induced Stat3 phosphorylation and epithelial to mesenchymal transition (EMT) -like features. Together, our data suggest that MGP-40 is involved in protection of BuMECs under stress conditions, inhibits cellular differentiation and induces EMT-like features. A schematic diagram depicting possible association of MGP-40 in various molecular pathways has been presented.

Testicular cytology indicates differences in Sertoli cell counts between "good freezer" and "poor freezer" bulls.

In artificial insemination, poor quality of semen unsuitable for cryopreservation and susceptibility of spermatozoa to cryodamage in crossbred bulls have been a matter of concern. Present study was designed to identify the testicular cytology indices that might be used to predict the semen quality and cryotolerance of spermatozoa in bulls. Based on the ejaculate rejection rate and sperm cryotolerance, bulls (Holstein Friesian X Tharparkar crossbred) were classified into either good (producing good quality semen with spermatozoa having good cryotolerance; n = 4) or poor (producing poor quality semen with spermatozoa having poor cryotolerance; n = 4). Testicular cytology was studied in all the 8 bulls using fine needle aspiration technique. Testicular cytology of good bulls and poor bulls differed significantly. The proportion of Sertoli cells was significantly higher in good bulls (25.3 ± 1.6) compared to poor bulls (11.0 ± 0.8). The Sertoli cell index was 46.1 ± 5.0 in good bulls while it was only 13.8 ± 1.3 in poor bulls. The cut off values, as determined using Receiver Operating Characteristics analysis, indicate that the bulls having testicular cytogram comprising of < 15.5% Sertoli cells, < 24.3 Sertoli cell index and > 4.0 spermatogenic cells to Sertoli cell ratio might be a poor bull in terms of semen quality and cryotolerance of spermatozoa. The proportion of Sertoli cells in the testicular cytology had positive (P < 0.05) relationship with semen quality and cryotolerance of spermatozoa.

Risk factors and impact of retained fetal membranes on performance of dairy bovines reared under subtropical conditions.

The risk factors and impact of retained fetal membranes (RFM) on productive and reproductive performance of crossbred cattle, Zebu cattle, and Murrah buffalos were evaluated using data spread over 12 years. Multivariable logistic regression model was used to identify risk factors and to quantify their odds ratio (OR). Overall incidence of RFM in crossbred cattle, Zebu cattle, and Murrah buffalos were 26, 16, and 13 %, respectively; and significant risk factors for RFM in crossbred cattle were abortion (OR = 3.9), dead calf (OR = 4.1), dystocia (OR = 4.3), pluriparity (OR = 1.5), and shorter gestation length (OR = 4.3). In Zebu cattle, abortion (OR = 4.0), dead calf (OR = 3.7), dystocia (OR = 3.9), lower birth weight of calf (OR = 1.6), and shorter gestation length (OR = 6.4) were significant risk factors for RFM. In Murrah buffalos, abortion (OR = 19.2), dead calf (OR = 4.4), dystocia (OR = 4.7), pluriparity (OR = 1.7), shorter gestation length (OR = 12.7), and calving during summer season (OR = 1.8) were the risk factors for RFM. Although the occurrence of RFM did not affect fertility parameters, a significant (P < 0.05) decrease in 305-day milk yield and total milk yield was observed in RFM-affected crossbred cattle. Taken together, it may be concluded that increased parity, abnormal calving, and short gestation length were the main risk factors for RFM in dairy bovine.

Bovine reproductive immunoinfertility: pathogenesis and immunotherapy.

Infertility is one of the primary factors for cattle reproduction in the present scenario. Reproduction-related immunoinfertility mainly involves immunization against the antigens related to reproductive hormones (LHRH, GnRH, Gonadal steroids, PGF2α and oxytocin), spermatozoa, seminal plasma and ovum. Anovulation, delayed ovulation, sperm immobilization, failure of fertilization, prolonged uterine involution, extended calving interval, prolonged post-partum estrus and reduced conception rate could be a result of immunoinfertility that occur due to the blockage of receptor site by antibodies formed against hormones, sperm and ovum. Immunoinfertility can be treated in the animal by giving sexual rest to females, by using various reproductive technologies such as in-vitro fertilization, gamete intra fallopian tube transfer, and intracytoplasmic sperm injection, sperm washing and by treating the animals with immunomodulators such as LPS, Oyster glycogen, etc. This review summarizes the different causes of bovine reproductive immunoinfertility and amelioration strategies to overcome it.

New dimensions on maternal and prepubertal nutritional disruption on bull fertility: A review.

Earlier, it was said that a bull is half of the herd because of its half contribution towards the genetic makeup in each subsequent generation. Nowadays, bulls are considered more than half of the herd because of the extensive use of frozen semen samples in artificial insemination. Bull's low fertility accounts for a major economic loss to livestock farmers. It is well known that fertility is a low-heritable trait governed by many factors such as genetics, epigenetics, climate, stress, and physical soundness. Apart from all these factors, the nutritional status of the bull also affects the semen quality. It has been seen that a bull given undernutrition at an early age is affected by androgen synthesis and semen quality. The nutrition given to the pregnant dam also affects the male progeny's postnatal semen quality. However, more studies are needed to elucidate the effect of periconception nutrition on the fertility of progeny as far as bulls are considered. This review focused on the effect of maternal undernutrition during the periconception period and undernutrition during the early growth phase of bull calves on the postnatal fertility of bulls.

The cryopreservation process induces alterations in proteins associated with bull sperm quality: The equilibration process could be a probable critical control point.

The present study quantitatively characterized the proteomic changes in bull spermatozoa induced by the cryopreservation process. We performed high-throughput comparative global proteomic profiling of freshly ejaculated (before cryopreservation), equilibrated (refrigerated storage; during cryopreservation), and frozen (ultralow temperature; after cryopreservation) bull spermatozoa. Using the liquid chromatography-mass spectrometry (LC-MS/MS) technique, a total of 1,692, 1,415, and 1,286 proteins were identified in fresh, equilibrated, and cryopreserved spermatozoa, respectively. When the proteome of fresh spermatozoa was compared with equilibrated spermatozoa, we found that 166 proteins were differentially expressed. When equilibrated spermatozoa were compared with cryopreserved spermatozoa, we found that 147 proteins were differentially expressed between them. Similarly, we found that 156 proteins were differentially expressed between fresh and cryopreserved spermatozoa. Among these proteins, the abundance of 105 proteins was lowered during the equilibration process itself, while the abundance of 43 proteins was lowered during ultralow temperature preservation. Remarkably, the equilibration process lowered the abundance of sperm proteins involved in energy metabolism, structural integrity, and DNA repair and increased the abundance of proteins associated with proteolysis and protein degradation. The abundance of sperm proteins associated with metabolism, cGMP-PKG (cyclic guanosine 3',5'-monophosphate-dependent protein kinase G) signaling, and regulation of the actin cytoskeleton was also altered during the equilibration process. Collectively, the present study showed that the equilibration step in the bull sperm cryopreservation process was the critical point for sperm proteome, during which a majority of proteomic alterations in sperm occurred. These findings are valuable for developing efficient protocols to minimize protein damage and to improve the quality and fertility of cryopreserved bull spermatozoa.

RNA-Seq analysis reveals functionally relevant coding and non-coding RNAs in crossbred bull spermatozoa.

Sperm, which are believed to be transcriptionally and translationally inactive, synthesize RNA and proteins before there is gradual disappearance of the ribosome during chromatin compaction. Sperm transfer several functionally relevant transcripts to the oocyte, controlling maternal-zygotic transition and embryonic development. The present study was undertaken to profile and analyze sperm transcripts comprehensively using Next Generation Ribonucleic acid sequencing technology in Holstein Friesian x Tharparkar crossbred bulls. The results from global transcriptomic profiling revealed transcripts for 13,814 genes; of which 431 transcripts were expressed with >1 FPKM and 13,383 transcripts were expressed with >0 or <1 FPKM. The abundant mRNA transcripts of crossbred bull sperm were PRM1 and HMGB4. Gene ontology of transcripts with>1 FPKM revealed there was a major involvement in the structural constituent of ribosomes and translation. Results from pathway enrichment indicated the connection between ribosome, oxidative phosphorylation and spliceosome pathways and the transcripts of crossbred bull spermatozoa. The transcriptional abundance of selected genes, validated using RT-qPCR, indicated significant variations between bulls. Collectively, it may be inferred that the transcripts in crossbred bull sperm were heavily implicated in functions such as the structural constituent of ribosomes and translation, and pathways such as ribosome, oxidative phosphorylation and spliceosome. Further studies using larger sample sizes are required to understand the possible implications of transcriptomic variations on semen quality and fertility.

Modification of French mini-straw plug position for cryopreservation of small doses of bull sperm.

Greater than optimal diluting of semen for producing a large number of doses containing relatively small numbers of sperm can lead to compromised quality of sperm, post-thawing. In the present study the French mini-straw plug position was modified and the effect of re-positioning was evaluated on the quality of sperm after thawing subsequent to cryopreservation of small doses of sperm. Four types of mini-straws were used based on the position of cotton plug including no plug displacement (Type 1; Manufacturers location for plug-placement in 0.25 mL French mini-straws), and Type II, III, and IV with re-positioning the cotton plug being 2.5, 5, and 7.5 cm, respectively, further from the manufacturer's placement location. Each ejaculate was proportioned into four Aliquots (I, II, III, and IV) and diluted to 80, 60, 40, and 20, million sperm/mL, respectively. Aliquot I was placed in all types of straws, while Aliquots II, III, IV were placed only in Type I straws. Semen straws were equilibrated, cryopreserved and sperm kinetic and functional variables were evaluated post-thawing. The results indicate that in Aliquots III and IV there were lesser (P < 0.05) values for sperm kinetic and function variables compared with sperm from Aliquot I (i.e., unmodified mini-straw). In conclusion, cryopreservation of small doses of sperm in modified French mini-straws resulted in acceptable values for kinetic and function variables, post-thawing.

Transcriptional abundance of type-1 endocannabinoid receptor (CB1) and fatty acid amide hydrolase (FAAH) in bull spermatozoa: Relationship with field fertility.

A highly sophisticated endogenous cannabinoid system (ECS) has been shown to play a crucial role in controlling sperm functions and fertility in men. In the present study, we report the differences in the expression level of components of ECS [type-1 endocannabinoid receptor (CB1) and fatty acid amide hydrolase (FAAH)] in spermatozoa from bulls with different field fertility ratings. Cryopreserved spermatozoa from crossbred cattle bulls (n = 40) were utilized for the study. The bulls were classified into high-, medium- and low-fertile bulls based on field conception rates. Sperm viability, capacitation status and protamine deficiency were assessed. Spermatozoa RNA was isolated from all the bulls, cDNA was synthesized and quantitative real time PCR was carried out to study the transcriptional abundance of CB1 and FAAH genes. Sperm viability was lower and capacitation was higher (p < 0.05) in low fertile bulls compared to medium and high fertile bulls. The expression level of CB1 gene was significantly (p < 0.05) lower in spermatozoa from low and medium fertile bulls compared to high fertile bulls. The expression of CB1 gene was 21.07 and 4.23 times greater in high and medium fertile bulls, respectively compared to low fertile bulls. The correlation between CB1 gene expression and field conception rate of bulls was positive and significant (r = 0.57; p < 0.001). Unlike CB1 receptors, FAAH gene expression was similar among high, medium and low fertile bulls. The correlation of FAAH expression with bull conception rate was positive but not significant. It was concluded that the transcriptional abundance of type-1 endocannabinoid receptor (CB1) was positively and significantly related to bull fertility.

Incubation of spermatozoa with Anandamide prior to cryopreservation reduces cryocapacitation and improves post-thaw sperm quality in the water buffalo (Bubalus bubalis).

Anandamide (AEA), an endocannabinoid, has been shown to reduce capacitation and acrosomal exocytosis in human spermatozoa. Because buffalo spermatozoa are highly susceptible to cryopreservation induced damage, AEA was assessed as to whether it could protect spermatozoa from cryo-damage. Six ejaculates from six Murrah buffalo bulls (total 36 ejaculates) were utilized for the study. Each ejaculate was divided into four aliquots; spermatozoa in Aliquot 1 were extended in Tris-Citrate-Egg Yolk and frozen as per the standard protocol. Spermatozoa in Aliquots 2, 3 and 4 were incubated with AEA at 1 nM, 1 µM and 10 µM, respectively in Tris-Citrate extender for 15 min at 37 °C before cryopreservation. Cryopreserved spermatozoa were thawed at 37 °C for 30 s before assessment of sperm motility, membrane integrity, capacitation, acrosome reaction, mitochondrial membrane potential (MMP) and lipid peroxidation status. The proportion of motile and membrane intact spermatozoa were greater (P < 0.05) with use of 1 µM AEA incorporated group compared with other groups. The proportion of un-capacitated and acrosome intact spermatozoa was greater (P < 0.05) with use of 1 or 10 µM of AEA compared with the other groups. When compared to the control group, use of 1 µM AEA resulted in a greater proportion of spermatozoa with high MMP (P < 0.05). There was no significant difference in the lipid peroxidation status of spermatozoa among any of the four groups. It was inferred that the protective role of AEA during cryopreservation of buffalo spermatozoa was dose dependent and incubation of spermatozoa with AEA at 1 µM concentration prior to cryopreservation reduced cryo-capacitation and improved post-thaw sperm quality in buffalo.