Kisspeptin-10 in cryodiluent improves the post-thaw quality of Nili-Ravi buffalo (Bubalus bubalis) bull spermatozoa.

Effects of kisspeptin-10 as antioxidant in cryodiluent were evaluated on post-thaw quality of buffalo spermatozoa. Qualified semen samples from five bulls were pooled, divided into five aliquots and extended in Tris-citric acid cryodiluent containing differential doses of kisspeptin-10 (5, 10, 15, and 20 µmol L-1 and negative control. Extended sperm suspension was cooled to 4°C, packaged in 0.54 ml straws and cryopreserved. At post-thawing, catalase (unit mg-1 ), peroxidase (unit mg-1 ) and reduced glutathione (µmol L-1 ) levels were highest (p < 0.05) with 20 µmol L-1 of kisspeptin-10 as compared to negative control. Moreover, lipid peroxidation (nmol L-1  min-1  mg protein-1 ) level was lowest (p < 0.05) with 20 µmol L-1 of kisspeptin-10. Sperm progressive motility (%), rapid velocity (%) and kinematics were higher (p < 0.05) with 15 and 20 µmol L-1 of kisspeptin-10 as compared to negative control. Supra-vital plasma membrane integrity (%), viable sperm with intact acrosome (%) and DNA integrity (%) were improved (p < 0.05) with all doses of kisspeptin-10 as compared to negative control. It was concluded that the addition of 15 and 20 µmol L-1 kisspeptin-10 in cryodiluent ameliorated the overall frozen-thawed quality parameters of Nili-Ravi buffalo spermatozoa.

Validation of double freezing protocol for Beetal buck (Capra hircus) spermatozoa.

The study aimed to validate the double versus single freezing protocol for Beetal buck (Capra hircus) spermatozoa in tris-citric acid (TCA) based extender both in terms of quality and fertilization potential. Computer-assisted sperm motion and kinematic (CASA) variables, i.e. total (%), and progressive motilities (TM and PM, %) and rapid velocity (RV, %), average path (VAP, µm/s), straight line (VSL, µm/s) and curved line velocities (VCL, µm/s), straightness, (VSL/VAP, %) and linearity, (VSL/VCL, %) as well as supra-vital plasma membrane integrity (SV-PMI, %), mitochondrial membrane potential (MMP, %), viable/intact acrosome (V-IACR, %) and DNA integrity (DNA-I, %) had significantly greater values (p < .05) during single freeze-thawing as compared with the double freeze-thawing at 0, 30, 90, 150 and 210 days, respectively. All CASA and other assays alone did not show significant differences (p > .05) between both freeze-thaw cycles at all treatment durations, respectively. No statistical significance (p > .05) was observed for the in vivo fertility between single (n = 84/141 = 59.72%) and double freeze-thawing (n = 72/136 = 52.9%) cycles, respectively. In conclusion, sperm motion, kinematics, plasma membrane, acrosome, mitochondria and DNA integrities and in vivo fertility are acceptable after the double freezing protocol despite being lower than after one freeze cycle in Beetal buck.

Approaches of estrous synchronization in sheep: developments during the last two decades: a review.

The objective of the current review was to summarize the protocols used for estrous synchronization in ewes during the last two decades. Progesterone (P4) is a major hormone used in most protocols. P4 in the form of a controlled internal drug releasing (CIDR) device, medroxyprogesterone acetate (MAP), and fluorogestone acetates (FGA) has been used for estrous synchronization. Also, gonadotropins such as equine chorionic gonadotropin (eCG) and gonadotropin-releasing hormone (GnRH) are often administered at the end of P4-based protocols to improve fertility. Moreover, the administration of prostaglandins (PG) and ram effects have been used for estrus induction and synchronization of ewes. The findings of previous studies indicate that the outcome of administering various synthetics P4 analogues (CIDR, MAP, and FGA) in ewes is comparable in terms of estrous synchronization/induction. The supplementation of P4-based protocols with eCG, however, improves the estrus response and pregnancy rate during breeding and non-breeding season. On the other hand, PG is effective for successful estrous synchronization during the breeding season only. Often, two injections of PG are administered either 11 or 14 days apart along with P4-based protocols to lyse ovine corpus luteum (CL) when it is receptive to PG i.e., 3 days post-ovulation. Alternatively, the "ram effect" has been shown to improve the efficacy of P4-based protocols and can be used as an alternative to eCG in ewes. The current review describes the methods of synchronization and their outcomes during breeding and a non-breeding season in ewes.

Quercetin in semen extender improves frozen-thawed spermatozoa quality and in-vivo fertility in crossbred Kamori goats.

This study investigated the antioxidant effect of quercetin-treated semen on frozen-thawed spermatozoa quality and in-vivo fertility in crossbred Kamori goats. In total, 32 ejaculates from four fertile bucks were diluted in Tris-based egg yolk extender with varying levels of quercetin (0, 1, 5, 10, and 15 µM). Qualified semen samples were pooled and frozen in French straws. The results revealed that the addition of quercetin in the semen extender increased (p < 0.05) frozen-thawed sperm total motility (TM), progressive motility (PM), rapid velocity (RV), average path velocity (VAP), straight line velocity (VSL), curvilinear velocity (VCL), and amplitude of lateral head (ALH) displacement in contrast to the control group. Quercetin supplementation had no effect on beat cross frequency (BCF), straightness (STR), and linearity (LIN) (p > 0.05). Quercetin showed significantly higher (p < 0.05) plasma membrane and acrosome integrity and viability (p < 0.05) of spermatozoa in contrast to the control group. Quercetin in the semen extender significantly increased (p < 0.05) superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), and total antioxidant capacity (TAC) levels while reduced (p < 0.05) the contents of total oxidant status (TOS) and malondialdehyde (MDA), which were in contrast to the control group. Ultrasound results revealed that 24 out of 30 (80%) goats were found pregnant when semen was treated with 5 µM quercetin while the control group showed 18 out of 30 (60%) animals were pregnant. Thus, the study concluded that 5 µM quercetin-treated semen was found to be efficient, showed increased antioxidant status, and reduced oxidant production, leading to improved spermatozoa quality and in-vivo fertility in goats.

Effects of vitamins C and E in tris citric acid glucose extender on chilled semen quality of Kail ram during different storage times.

Mammalian sperm cells are highly vulnerable to lipid peroxidation by free radicals. Antioxidants such as vitamin E, and vitamin C neutralize the activity of free radicals and protect the sperm from reactive oxygen species. The present study was conducted to investigate the effects of vitamin C, vitamin E, and their combination in a Tris-based extender on the semen quality of Kail Ram. Semen samples from five mature Kail rams were collected in this study. The semen samples were diluted by Tris-glucose-egg yolk. Diluted semen samples were divided into four parts. The first part was added with 1 mg/ml of vitamin C, the second part was added with 1 mg/ml of vitamin E and the third part was added with both vitamin C and E, in combination with a dose of 1 mg/ml. The fourth part was considered as control without any addition. The diluted semen samples were cooled gradually and preserved at 5˚C for three days. Sperms in chilled diluted semen samples were examined for motility, viability, and plasma membrane integrity every 24 h for three days (72 h). Present study results showed significant (P < 0.05) effects of vitamins C, E, and their combination on some parameters such as motility, straightness of average special path, linearity of the curvilinear trajectory, and beat-cross frequency. However, there was no significant (P < 0.05) effect of storage duration and antioxidants (vitamin C, E, and their combination) on viability, sperm plasma membrane integrity, and some CASA parameters. From the present study, it could be concluded that the supplementation of vitamins C, and E, and their combination do not enhance the life span and quality of semen in Kail ram during liquid storage at 5 °C.

A systematic review and meta-analysis of spermatozoa cryopreservation, in vitro and in vivo fertility practices in water buffalo.

We explored different aspects of buffalo spermatozoa during cryopreservation. The meta-data comprised of 285 studies, published from January 2008 to March 2020. A free web tool CADIMA as well as PRISMA 2009 Flow Diagram were used for carrying out this study. The inter-reviewer agreement among studies allocated was satisfactory for criteria A (selection bias), B (performance bias), C (detection bias) and D (attrition bias), respectively. India led the percent (%) research ladder with 34.4, followed by Pakistan (29.5), Egypt (12.3), Iran (7.7), Italy (5.6), Indonesia (3.2), China (2.1), Brazil (1.4), Thailand (1.1), Philippines and Bulgaria (0.7 each), Saudi Arabia, Turkey, Vietnam, and USA (0.4 each). Among four categories of studies, Group-1 evaluated only supplements/additives/media in the freezing semen extender (n = 191/285; 67.02%); Group-2 conducted in vivo fertilization (n = 62/285; 21.75%) and Group-3 conducted in vitro fertilization/ cleavage rate/penetration rate/ blastocyst yields (n = 28/285; 9.82%) with their specific cryodiluents/media, respectively. Group-4 conducted different experimental supplements/additives/media and carried out both in vitro and in vivo fertilization simultaneously (n = 4/285; 1.40%). Conventional spermatozoa cryopreservation was reported by 51.9% studies followed by programmable fast freezing by 20.7% studies. A few leading extender types included BioXcell (3.9%); Soyamilk-skim (3.5%); and Andromed (2.1%). The study also describes French straws for semen filling, cooling temperatures, extension time, equilibration time, cryopreservation stages, thawing temperatures, seasons, thawing time, and stains used during semen evaluation assays. The study concludes that the research on spermatozoa cryopreservation of buffalo is largely conducted at quality level and a need of applying these findings for evaluation of fertility potential (in vivo and in vitro) is indispensable for effective genetic improvement.

Cryoprotection synergism between glycerol and dimethyl sulfoxide improves the mitochondrial transmembrane potential, plasmalemma, acrosomal and DNA integrities, and in vivo fertility of water buffalo (Bubalus bubalis) spermatozoa.

The objective of the study was to devise a cryoprotection synergism between glycerol and dimethyl sulfoxide (DMSO) for water buffalo spermatozoa. Additionally, the effect of best evolved concentrations of glycerol and DMSO in extender was assessed on in vivo fertility of buffalo spermatozoa. Ejaculates (n = 30) were equally distributed into five aliquots; first aliquot was diluted at 37 °C in extender having 7 % glycerol (control); second aliquot was diluted at 37 °C as well as at 4 °C in extender having 3.5 % DMSO (Group 1); third aliquot was diluted at 37 °C in extender having 3.5 % glycerol and then at 4 °C in extender having 3.5 % DMSO (Group 2); fourth aliquot was diluted at 37 °C in extender having 3.5 % DMSO and then at 4 °C in extender having 3.5 % glycerol (Group 3); fifth aliquot was diluted in extenders having 1.75 % glycerol and 1.75 % DMSO at 37 as well as at 4 °C (Group 4). At post thawing, sperm progressive motility (%), rapid velocity (%), average path velocity (µm/s), curved line velocity (µm/s), in vitro longevity (%), structural and functional integrity of plasmalemma (%), mitochondrial transmembrane potential (%) and viable sperm with intact acrosome (%) were higher (P < 0.05) in Group 4 compared to other treatment groups and control. Regarding sperm DNA integrity (%); it was higher (P < 0.05) in Group 4 compared to Group 1, 3 and control. The in vivo fertility (%) of buffalo spermatozoa was significantly higher with Group 4 compared to control (69.45 vs. 59.81). In conclusion, synergism exists between glycerol and DMSO (Group 4) in improving the quality and in vivo fertility of cryopreserved water buffalo spermatozoa.

Trehalose improves semen antioxidant enzymes activity, post-thaw quality, and fertility in Nili Ravi buffaloes (Bubalus bubalis).

Our objectives were to study the effect of trehalose in extender on (1) antioxidant enzymes profile during cryopreservation (after dilution, before freezing, and after thawing), (2) in vitro quality (after thawing), and (3) in vivo fertility of Nili Ravi buffalo (Bubalus bubalis) bull spermatozoa. Semen samples (n = 20) from four buffalo bulls were diluted in Tris-citric acid-based extender having different concentrations of trehalose (0.0, 15, 30, 45, and 60 mM) and frozen in French straws. At post dilution, profile of sperm catalase (U/mL) was higher (P < 0.05) in extenders containing 15, 30, and 45 mM of trehalose as compared to control. Although profiles of superoxide dismutase (U/mL) and total glutathione (µM) were higher (P < 0.05) in extenders containing 15 and 30 mM of trehalose as compared to control. At prefreezing, sperm catalase, superoxide dismutase, and total glutathione profiles were higher (P < 0.05) in all the treatment groups as compared to control. At post thawing, the profiles of catalase and total glutathione were higher (P < 0.05) in extender containing 30-mM trehalose as compared to other treatment groups and control. Whereas, profile of superoxide dismutase was higher (P < 0.05) in extenders containing 30, 45, and 60 mM of trehalose as compared to control and 15mM group. Post thaw total sperm motility (%) was higher (P < 0.05) in extender containing 30-mM trehalose as compared to control and 15 and 60-mM groups. Although sperm progressive motility (%), rapid velocity (%), average path velocity (µm/s), straight line velocity (µm/s), curvilinear velocity (µm/s), plasma membrane (structural and functional, %), acrosome (%), and DNA (%) integrity were higher (P < 0.05) in extender containing 30 mM trehalose as compared to other treatment groups and control. The fertility rates (61% vs. 43%) were higher (P < 0.05) in buffaloes inseminated with semen doses cryopreserved in extender containing 30 mM of trehalose than the control. It is concluded that addition of 30-mM trehalose in extender improves the semen antioxidant enzymes activity, post thaw quality, and fertility in Nili Ravi buffaloes.