In vitro evaluation of Labrador dog spermatozoa cryopreserved in Tris-citric acid-fructose buffer supplemented with different combinations of extracellular and intracellular cryoprotectants.

Aim of this study was to compare different combinations of penetrating intracellular CPAs, i.e., glycerol (G), ethylene glycol (EG), propylene glycol (PG), dimethyl formamide (DM), and methyl acetamide (MA) and extracellular [egg yolk (EY), egg yolk plasma (EYP), low-density lipoproteins (LDL), and coconut water (CW)] in Tris-citric acid-fructose buffer (T) for Labrador dog semen cryopreservation. The study was conducted in two parts, first trial was conducted to assess optimum glycerol concentration (5-7%) in TEY and equilibration time (ET, 2-4 hrs) for Labrador dog semen cryopreservation. Secondly, compatibility of 15% TEY, 15% TEYP, 13% TLDL, and 25% TCW with G, DMF, MA, D + M, EG, and PG was evaluated for in vitro sperm function tests. Decline in sperm attributes, i.e., motility, viability, plasma membrane integrity (PMI), and acrosome integrity (AI)) was significantly (p < 0.05) less in 7% TEY-G and 4 h compared to other concentrations and ET at post-thaw. There was significantly (p < 0.05) less decline in sperm attributes in TEY-G, TEYP-G, TLDL-G, TLDL-D, TLDL-EG, and TCW-D extenders compared to other combinations at post-thaw. However, these parameters were significantly (p < 0.05) high in TEY-G and TEYP-G compared to TEYP-D, TLDL-G, TLDL-D, TLDL-EG, and TCW-D extenders at post-thaw. However, decline in motility, viability, PMI, and AI was identical in these seven extenders. This study concluded that glycerol at a concentration of 7% in TEY and 4 h ET were optimum for successful cryopreservation and besides TEY-G, other combinations of protectants may be an alternative for canine semen cryopreservation.

Association of antioxidant defense system with semen attributes vis a vis fertility in exotic and indigenous chicken breeds.

The present study was designed to ascertain the association of antioxidant defense system with semen attributes and fertility in RIR (Rhode Island Red), PR (Punjab Red), RIR x local cross, Aseel and Kadaknath breeds. Based on sperm attributes, roosters of each breed were divided into two group i.e. G-I (exhibiting >50% sperm attributes) and G-II (exhibiting <50% sperm attributes). Motility, viability, membrane-, acrosome-, DNA-integrity and fertility differed significantly (p < 0.05) among the breeds, representing maximum in PR roosters and least in Aseel roosters. Values of sperm attributes and fertility rate were also significantly (p < 0.05) higher in G-I compared to G-II roosters in exotic as well as indigenous breeds. MDA content differed significantly (p < 0.05) in spermatozoa of five breeds. It indicated a least oxidant stress in PR and highest in Aseel. MDA concentration was significantly (P < 0.05) higher in G-II (28.36 ± 2.40-96.0 ± 6.4) than G-I (13.65 ± 1.81-52.22 ± 6.4) roosters in all breeds. Antioxidant enzyme activity varied significantly (p < 0.05) among the breeds and groups within the breeds. Significantly (p < 0.05) higher activity of four enzymes was evident in G-II as compared to G-I roosters irrespective of the breed. A moderate to strong negative correlation was perceived among LPO, SOD, GPX, catalase, GRE and sperm attributes/fertility rate. A moderate to strong positive correlation (0.21-0.92) among LPO and antioxidant enzymes revealed that with the increase in LPO, antioxidant enzymes increase too and vice versa in all breeds. Likewise, positive correlation between sperm attributes and fertility revealed that higher sperm attributes contribute to the high fertility of roosters. This is one of the first reports on complete set of antioxidant enzymes and oxidative stress in relation to sperm attributes and fertility in different five chicken breeds. A complete antioxidant enzyme system seems to modulate the oxidative stress, sperm attributes and fertility. It may be possible to use LPO as a fertility marker to select the roosters for breeding purpose in the chicken breeds.

Identification of sperm-specific proteins associated with high fertility of Punjab Red and RIR x Local cross roosters with SDS-PAGE and Immunoblotting.

The present study aimed at characterization of fertility-associated proteins in PR and RIR x Local roosters and was conducted on two generations of birds. Roosters were divided into high- (>50%) and low-fertility groups (<50%) based on sperm function tests and fertility rate in both the generations. Polyclonal antibodies were raised in rabbits against sperm proteins of first generation highly fertile roosters and tested for characterization of fertility-associated sperm proteins in the second generation of same roosters. IgG fraction against proteins (anti-SP IgG) was reacted with sperm proteins of both high and low fertile roosters of second generation on immunoblots. SDS-PAGE of sperm extracts of PR and RIR x Local cross breeds resulted in resolution of 12 and 23 proteins on 12% acrylamide gels and anti-SP IgG reacted only with 8 and 9 sperm proteins of PR and RIR x Local cross roosters on immunoblots. The SDS-PAGE and immunoblotting analysis also indicated a variation in sperm proteins among two breeds and high/low fertile roosters. It can be concluded that the selection of roosters on the based on proteins of 65/ 25; 70/ 46/ 30 kDa may be specifically associated with high fertility of PR and RIR x Local cross, respectively. The proteins 62 kDa (PR) and 40kDa (RIR x Local cross) may be specifically responsible for low fertility.

Characterization of fertility associated sperm proteins in Aseel and Rhode Island Red chicken breeds.

This study focused on characterization of fertility associated proteins in Aseel and RIR roosters and was conducted on two generations of birds. Roosters were divided into high (>50%) and low fertility groups (<50%) based on sperm function tests and fertility rate in both the generations. Polyclonal antibodies were raised in rabbits against sperm proteins of first generation highly fertile roosters and tested for characterization of fertility associated sperm proteins in the second generation of same roosters. IgG-fraction against proteins (Anti-SP-IgG) was reacted with sperm proteins of both high and low fertile roosters of second generation on immunoblots. Sperm proteins present in highly fertile roosters were further characterized by Mass Spectrometry (MS). Use of SDS-PAGE for evaluation of sperm extracts of Aseel and RIR breeds resulted in resolution of 16 and 10 proteins on 12% acrylamide gels. Anti-SP-IgG reacted with eight and ten sperm proteins of Aseel and RIR roosters on immunoblots. The SDS-PAGE and immunoblotting analysis also indicated a variation in sperm proteins among two breeds and high/low fertile roosters. The MS analysis indicated matching of 20, 30, and 20, 25 kDa proteins (associated with high fertility rate) of Aseel and RIR roosters with immunoglobulin kappa chain variable, phospholipase A2 (PLA2), hypothetical N332-08551 partial and cystatin like partial proteins with a top score of 41, 46, 52 and 43, respectively. Considering the function and importance of matching proteins in male reproduction, these proteins may be further explored as potential markers for fertility evaluation of Aseel and RIR roosters.

Effect of scrotal bifurcation on breeding soundness traits in Beetal bucks during summer season.

Relationship of scrotal bifurcation or splitness with breeding soundness traits was investigated in 15 Beetal bucks (17-20 months of age and 48.72 ± 1.57 kg mean BW). Breeding soundness traits of conjoined/unsplit (n = 6) scrotal bucks was compared with split (n = 9) scrotal bucks having lengthwise > 1 in. bifurcation. Two consecutive semen ejaculations per buck were collected at monthly interval during summer season (April to June 2018) using intact buck as teaser and sexual behavior was simultaneously recorded. Scrotal morphometry parameters, i.e., scrotal circumference, scrotal volume, scrotal skin thickness, testis length, width, and thickness were also recorded. Bucks with split scrotum had relatively more scrotal dimensions (scrotal circumference (P < 0.01), scrotal volume (P < 0.01), testis length (P < 0.01)) than conjoined scrotal bucks. Among various semen attributes, semen volume of first ejaculate (P < 0.01), second ejaculate (P < 0.05), mean semen volume (P < 0.01), total sperm count of first ejaculate, and mean sperm count of both ejaculates were more (P < 0.01) in split scrotal bucks. However, scrotal bifurcation had no influence on sexual behavior of bucks. It is concluded that Beetal bucks with split scrotum had relatively better breeding efficiency traits than conjoined scrotal bucks.

Clinical efficacy of intrauterine cephapirin benzathine administration on clearance of uterine bacteria and subclinical endometritis in postpartum buffaloes.

This study examined the effect of single IU administration of cephapirin on clinical recovery, clearance of uterine bacteria and reproductive performance of postpartum buffaloes with subclinical endometritis (SCE). Buffaloes (n = 86) at 35 days postpartum (DPP) with >10% polymorphonuclear (PMN) cells in endometrial cytosmears were designated as positive (SCEP, n = 29), and buffaloes with ≤10% PMN cell were designated as negative (SCEN, n = 57) for SCE. Out of 29 positive buffaloes, 15 were administered a single intrauterine dose of cephapirin benzathine on 40 DPP (SCEP-CB), while the remaining 14 animals were kept as untreated control (SCEP-C). All animals were observed regularly for oestrous signs and were again subjected to cytobrush sampling on the first postpartum (FPP) oestrus. Buffaloes positive for SCE at 35 DPP were later considered "recovered" if their PMN cells dropped to ≤5% on the FPP oestrus. Presence of Escherichia coli, Arcanobacterium pyogenes and Fusobacterium necrophorum in uterus was detected based upon PCR amplification of genes related to bacteria-specific virulence factors. A total of 66.7% of SCEP-CB group buffaloes recovered as compared to 28.6% in SCEP-C (χ2  = 4.21; p < 0.05). Rate of bacterial clearance did not differ between treated (38.5%) and untreated buffaloes (8.3%) (χ2  = 1.67; p > 0.05). The median days to first service did not differ significantly (p > 0.05) among the three groups, whereas cephapirin administration reduced (p < 0.05) the days open by 14 days in SCEP-CB compared to SCEP-C buffaloes. SCEP-CB buffaloes were as likely to conceive as SCEN, whereas SCEP-C had 0.28 hazard ratio for pregnancy. In conclusion, a single treatment with cephapirin benzathine at 40 DPP improved the reproductive performance of buffaloes with subclinical endometritis.

Homology between cattle bull sperm and bacterial antigenic proteins viz a viz possible role in immunological infertility.

This study was carried out to investigate the possible presence of identical sperm and bacterial antigens which may cause similar antisperm antibody production leading to lower fertility. Cross-reactive antigens of cattle bull spermatozoa and different bacteria including Escherichia coli (E. coli), Bacillus sp., and Staphylococcus sp. were characterized by immunoblotting and mass fingerprinting. Significant cross-reactivity was obtained for 75, 72, 44, 40, 33, 30, 25, 18, 14 kDa proteins with purified IgG of calves, heifers and cows between spermatozoa and the studied bacteria. Significantly (p < 0.05) matched cross-reactive 40/33/30 kDa sperm, 33 kDa Staphylococcus sp/Bacillus sp and 40/25 kDa E. coli proteins were analyzed. Mass fingerprinting of 40/33/30 kDa (spermatozoa); 40/25 kDa (E. coli) and 33 kDa (Bacillus/Staphylococcus) proteins revealed their matching with vitellogenin-1-like/mitochondrial malate dehydrogenase 2, NAD/acrosin-binding protein isoform XI; outer membrane insertion signal domain/spore coat protein and glyceraldehyde-3-phosphate dehydrogenase, respectively. Acrosin-binding protein isoform X1 and mitochondrial malate dehydrogenase 2, NAD contributes to the capacitation of spermatozoa. Spore coat protein; glyceraldehyde-3-phosphate dehydrogenase of E. coli; Bacillus/Staphylococcus are 37.6% and 39.01% identical to acrosin-binding protein isoform X1; mitochondrial malate dehydrogenase 2, NAD of cattle bull spermatozoa. It can be interpreted from these observations that cross-reacting antibodies developed against 33/30 kDa sperm proteins and 25, 33 kDa bacterial proteins in cows may affect the functional activity of spermatozoa leading to delayed fertility in heifers and cows.

Seminal Plasma Heparin Binding Proteins Improve Semen Quality by Reducing Oxidative Stress during Cryopreservation of Cattle Bull Semen.

Heparin binding proteins (HBPs) are produced by accessory glands. These are secreted into the seminal fluid, bind to the spermatozoa at the time of ejaculation, favour capacitation, acrosome reaction, and alter the immune system response toward the sperm. The present study was conducted with an objective to assess the effect of purified seminal plasma-HBPs (SP-HBPs) on cross bred cattle bull sperm attributes during two phases of cryopreservation: Pre freezing and freezing-thawing. SP-HBPs were purified from pooled seminal plasma by heparin affinity chromatography. Three doses of SP-HBPs i.e. 10, 20, 40 µg/mL semen were standardized to find out the optimum dose and 20 µg/mL was found to be an optimum dose. Semen as such and treated with SP-HBPs was diluted with sodium citrate-egg yolk diluter and cryopreserved as per the standard protocol. Sperm parameters i.e. motility, viability, Hypo-osmotic swelling test (HOST), acrosome damage, in vitro capacitation and lipid peroxidation were evaluated in SP-HBP treated and untreated (control) semen at both phases of cryopreservation. A considerable variation in percent sperm motility, viability, membrane integrity (HOST), acrosome damage, acrosome reaction and lipid peroxidation was observed at both phases among the bulls irrespective of the treatment. Incubation of neat semen with 20 µg/mL SP-HBP before processing for cryopreservation enhanced the average motility, viability, membrane integrity by 7.2%, 1.5%, 7.9%, and 5.6%, 6.6%, 7.4% in pre-frozen and frozen-thawed semen in comparison to control. There was also an average increase of 4.1%/3.9% in in vitro capacitation and acrosome reaction in SP-HBPs-treated frozen-thawed semen as compared to control. However, binding of SP-HBPs to the sperm declined acrosome damage and lipid peroxidation by 1.3%/4.1% and 22.1/32.7 µM/10(9) spermatozoa in SP-HBP treated pre-frozen/frozen-thawed semen as compared to control, respectively. Significant (p<0.05) effects were observed only in motility, HOST and in vitro acrosome reaction. It can be concluded that treatment of neat semen with SP-HBPs before cryopreservation minimized the cryoinjury by decreasing the generation of reactive oxygen species.

Manganese provides antioxidant protection for sperm cryopreservation that may offer new consideration for clinical fertility.

Reactive oxygen species (ROS) are generated by sperm metabolism. While, ROS are required for maturation, capacitation and acrosome reaction, they also modify many peroxidable cellular compounds. There is production of ROS during cryopreservation and frozen spermatozoa are highly sensitive to lipid peroxidation (LPO). Antioxidants exert a protective effect on the plasma membrane of frozen bovine sperm preserving both metabolic activity and cellular viability. Manganese (Mn(++)) is proved to be a chain breaking antioxidant in biological system. Therefore, we examined the role of (Mn(++)) during cryopreservation of cattle bull semen. Semen was divided into four parts and cryopreserved in egg-yolk-citrate extender + glycerol (EYC-G), EYC-G + 100 microM of Mn(++), EYC-G + 150 microM of Mn(++) and EYC-G + 200 microM of Mn(++). After four hours of cooling and 24 hrs of freezing, the spermatozoa were examined for percentage motility, Hypo-osmotic swelling (HOS), LPO and protein leakage. Addition of manganese to the semen during cryopreservation showed a protective effect and accounted for an increase in semen quality parameters [percentage motility, HOS percent and decrease in malondialdehyde (MDA) production and protein leakage]. The effect of manganese on motility and HOS was non-significant (p < 0.05) in cooled spermatozoa but significant with 150 microM of Mn(++) in frozen-thawed spermatozoa. MDA production and protein leakage decreased to a significant and maximum level (p < 0.05) on addition of 200 microM of manganese. The addition of manganese to EYC-G dilutor will improve the quality/fertility of semen, which will result in improvement of in vitro fertilization and artificial insemination success rate.

Response related enzymatic changes in ovaries of superovulated mice.

Adult female mice were superovulated with PMSG followed by HCG and 140 blastocysts and 69 morulae were recovered from 24 mice. On the basis of the response, mice were divided into six groups; non responders, 1-5, 6-10, 11-20, 21-30 and >30 embryos. The ovaries of the animals were pooled group wise, homogenized in PBS (pH 7.4) and after centrifugation for 10-15 minutes, the supernatant was analyzed for the enzymes, guanine oxaloacetate transaminase (GOT), guanine pymvate transaminase (GPT), acid phosphatases (ACP) and alkaline phosphatases (AKP). Acid and alkaline phosphatase activities did not show any variation in relation to response to superovulation but GOT and GPT showed significantly increased activity in response to induction of superovulation. A statistically significant positive correlation was found between GOT and GPT activities and the superovulatory response in mice.