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The trial was performed on 18 multiparous Mediterranean buffaloes, pre-synchronized by a double injection of 0.524 mg of PGF2α. Animals were divided into two groups (A and B), that underwent two superovulation (SO) treatments, starting 8 days after estrus (day 0): Group A (n=9) received a 4-days double daily decreasing dosage of 400 mg NIH-FSH-P1 (Folltropin®, Bio98, Italy). Group B (n=9) received the same treatment as Group A for the first 2 days, and a single injection of 1000 IU of eCG (Ciclogonina®, Fortdodge, Italy). On day 10 all buffaloes received a double injection of 0.524 mg of PGF2α to induce luteolysis. Ultrasound follicular development was assessed daily on each buffalo from the start of the SO treatments until 48 hours after estrus detection to record the total number of the follicles, grouped into 3 categories according to their diameter: small (between 0.20 and 0.50 cm), medium (between 0.51 and 1.00 cm) and large (more than 1.01 cm). The statistical analysis was carried out by using the chi-square test and the analysis of variance. Buffaloes in Group B showed a higher (P

Most systems for producing bovine embryos in vitro use glucose as an energy source despite putative toxic effects. Glucose has a selective embryotoxicity towards female embryos, due to the higher expression of the X-linked glucose-6-phosphate dehydrogenase gene (Kimura et al. 2005 Mol. Reprod. Dev. 72, 201–207). Recently, the replacement of glucose with citrate and myo-inositol in SOF medium supplemented with 5% bovine serum (BS) increased the percentage of female embryos (Rubessa et al. 2011 Theriogenology 76, 1347–1355). Serum also affects the sex ratio of in vitro-produced (IVP) bovine embryos, favoring the male gender (Gutierrez-Adan et al. 2001 Theriogenology 55, 1117–1126). The aim of this work was to evaluate the effect of glucose replacement with myo-inositol during in vitro culture, in the presence of either BS or BSA, on bovine embryo sex ratio. Abattoir-derived oocytes (n = 1164, over 4 replicates) were matured and fertilized in vitro as previously described (Rubessa et al. 2011). After 20 to 22 h of gametes co-incubation, zygotes were denuded and cultured for 7 days in SOF with: group A) 0.34 mM trisodium citrate + 2.77 mM myo-inositol + 5% BS (n = 287); group B) 0.34 mM tri-sodium citrate + 2.77 mM myo-inositol + 8 mg mL–1 BSA(n = 290); group C) 1.5 mM glucose + 5% BS (n = 302) and group D) 1.5 mM glucose + 8 mg mL–1 BSA (n = 285). Representative samples of blastocysts produced in each group (n = 96, 58, 99, and 70, respectively in groups A, B, C, and D) were sexed by PCR as previously described (Rubessa et al. 2011). Differences among groups in blastocyst yields were analyzed by ANOVA. The percentages of female embryos were analyzed by chi-square test. Blastocyst rates in group C were lower (28.1%) than those recorded in groups A, B, and D (35.9, 41.0 and 36.1%, respectively; P < 0.01). A higher (P < 0.05) percentage of female embryos was observed in group A (61.5%) compared to group C (45.5%), with intermediate values in groups B (51.7%) and D (60.0%). Therefore, the replacement of glucose with citrate and myo-inositol favored the development of female embryos in the presence of BS but was ineffective in the presence of BSA. Furthermore, when glucose was the energy source, a tendency to greater incidence of female embryos was observed when the medium was supplemented with BSA rather than BS (P = 0.06). As a small amount of glucose is present in the BS, we hypothesize an additional glucose-dependent toxic effect on female embryos in group C. However, we cannot rule out that other factors present in the BS may interact with the energy source, playing a role in determining the sex ratio. Furthermore, the shift in sex ratio in favor of males or females embryo can be due to a better development of embryo of one sex, or to the delayed development or degeneration of embryos of the other sex. In conclusion, these results suggest that manipulating the metabolic profile of the embryos during culture may have an impact on both blastocyst production and sex ratio.

In buffalo, in vitro embryo production (IVEP) technology is the best tool to improve the genetic merit through the maternal lineage. A major limitation of IVEP technology in buffalo species is the poor cryotolerance of the embryos, likely due to their high lipid content (Gasparrini 2002 Theriogenology 57, 237–256). It was previously demonstrated that supplementing bovine culture media with L-carnitine, a cofactor of β-oxidation, improves in vitro embryo development (Sutton-McDowall et al. 2012 Theriogenology 77, 1632–1641). The aim of this work was to evaluate whether L-carnitine supplementation during in vitro culture (IVC) improves blastocyst development and cryotolerance of in vitro produced buffalo embryos. After a preliminary dose response trial, we selected the concentration of 0.25 mM for the experiment. Cumulus–oocytes complexes (n = 288, over 4 replicates), recovered from slaughtered animals, were matured and fertilized in vitro according to our standard procedures (Gasparrini et al. 2006 Theriogenology 65, 275–287). On Day 1 (Day 0 = IVF), zygotes were cultured in SOF supplemented with 8 mg mL–1 BSA, in the absence (control, n = 143) or presence of 0.25 mM L-carnitine (n = 145). In vitro culture was carried out at 38.5°C under 5% CO2, 7% O2, and 88% N2. Cleavage rate was evaluated on Day 5, when the cleaved embryos were transferred into fresh medium for further 2 days. On Day 7 after IVF, embryo outcome was assessed and all the embryos were vitrified by cryotop in 16.5% ethylene glycol, 16.5% dimethyl sulfoxide (DMSO), and 0.5 M sucrose (De Rosa et al. 2007 Ital. J. Anim. Sci. 6(Suppl 2), 747–750). The resistance to cryopreservation was evaluated by assessing the survival rate, on the basis of morphological criteria, after 24 h culture. Data were analyzed by chi-square test. No differences were found in cleavage rates between the control (81.5%) and the L-carnitine group (78.8%). The blastocyst yields (calculated in relation to the cleaved embryos) were not significantly influenced by the L-carnitine treatment (40.2 and 52.9%, in the control and the L-carnitine groups, respectively). However, buffalo embryos cultured in the presence of L-carnitine showed an increased resistance to cryopreservation, as indicated by the higher survival rates recorded after 24 h culture (78.7 and 96.4%, in the control and the L-carnitine groups, respectively; P < 0.01). In conclusion, these results demonstrated that L-carnitine supplementation of culture medium improves the resistance to cryopreservation of in vitro produced buffalo embryos. We speculate that the increased cryotolerance observed in the presence of L-carnitine may be due to a better utilization of the endogenous lipid stores, resulting in improved embryo quality.

Despite the great improvement of in vitro embryo production (IVEP) efficiency recorded over the years in cattle, the in vitro produced (IVP) embryos are still less viable and resistant to cryopreservation than their in vivo counterparts. One of the major factor impairing in vitro embryo development is oxidative stress. Resveratrol is an important antioxidant polyphenolic compound found in several vegetal sources, that contributes to red wine’s beneficial effects on the prevention of human cardiovascular disease. Recently, the interest in resveratrol has increased exponentially following the major findings that this molecule has positive effects on cancer chemoprevention, cardioprotection, inflammatory processes, several aspects of metabolism, leading to increased lifespan of various organisms from yeasts to vertebrates (Pirola et al. 2008 IUBMB Life 60, 323–332). A positive effect of resveratrol on in vitro embryonic development was demonstrated in swine (Lee et al. 2010 J. Reprod. Dev. 56, 330–335). The aim of this study was to evaluate whether supplementation of culture medium with resveratrol improves in vitro blastocyst development and the embryo resistance to cryopreservation in cattle. A preliminary dose response trial indicated that the optimal concentration in the range tested (from 0.5 to 10 µM) was 0.5 µM, with evident toxic effects at concentration higher than 5 µM. Abattoir-derived oocytes (n = 581, over 5 replicates) were matured and fertilized in vitro according to our standard procedure (Rubessa et al. 2011 Theriogenology 76, 1347–1355). Twenty hours after IVF, presumptive zygotes were cultured in SOF medium, supplemented with 5% bovine serum, in the absence (control, n = 271) or presence of 0.5 µM resveratrol (n = 310) at 39°C under humidified air with 5% CO2, 7% O2, and 88% N2. On Day 7 (IVF = Day 0), embryo yields were assessed and the blastocysts (except the hatched blastocysts) were vitrified by cryotop in 16.5% ethylene glycol, 16.5% dimethyl sulfoxide (DMSO), and 0.5M sucrose (Rubessa et al. 2011). The resistance to cryopreservation was evaluated by assessing the survival rate, on the basis of morphological criteria, and hatching rate after 48 h culture. Data were analyzed by chi-square test. Resveratrol supplementation during culture did not affect either cleavage (69.1 v. 72.0%, in the control and resveratrol groups, respectively) or blastocyst yields (38.3 v. 36.3%, in the control and resveratrol groups, respectively). However, treatment with resveratrol increased the cryotolerance of IVP embryos, as indicated by higher survival rates (74.7 v. 88.4%, in the control and resveratrol groups, respectively; P < 0.05) and hatching rates (35.1 v. 53.8%, in the control and resveratrol groups, respectively; P = 0.06) at 48 h. In conclusion, these results demonstrated that resveratrol supplementation during culture improves the quality, and hence the resistance to cryopreservation, of IVP bovine embryos.

The aim of this work was to evaluate whether L-carnitine supplementation during IVC improves blastocyst development and cryotolerance of in vitro produced (IVP) buffalo embryos. Abattoir-derived cumulus-oocytes complexes (n=410, over 5 replicates) were matured and fertilized in vitro according to standard procedures (Gasparrini B et al. 2006 Theriogenology 65 (2), 275-287). On day 1 (Day 0 = IVF), zygotes were cultured in SOF supplemented with 8 mg/ml BSA, in the absence (control, n=165) or presence of L-carnitine (n=170) at a concentration (0.25 Mm) selected after a preliminary dose response trial. In vitro culture was carried out at 38.5°C under 5% CO2, 7% O2, and 88% N2. Cleavage and blastocyst rates (in relation to the cleaved embryos) were evaluated on Day 5 and 7, respectively. The blastocysts were vitrified by cryotop in 16.5% ethylene glycol, 16.5% DMSO and 0.5M sucrose and the survival rate, based on morphological criteria, was assessed after 24 h culture. Data were analyzed by Chi square test. Cleavage (81.3% vs 82.1%, in the control and carnitine groups) and blastocyst production (40.0% vs 47.6%, in the control and carnitine groups) were not affected by the treatment. The percentages of fast developing embryos (expanded and hatched blastocysts), i.e. those of better quality, were 17.0 and 23.5%, respectively. Interestingly, the embryos cultured with L-carnitine showed higher survival rates after 24 h culture (78.7% and 96.4%, in the control and carnitine groups, respectively; P

The improvement of fertility is an important contribution that applied scientific research can provide to buffalo breeding, especially in productive areas like the Campania region. Many studies have been conducted to improve the management of the female reproductive system, while insufficient are the information about the effect of the male on the reproductive efficiency in this species. Buffalo is a photoperiodic animal that tends to increase the reproductive activity during periods when daylight hours decrease. In particular, in the male, it is manifested by a reduction of libido when daylight hours increase. This causes a reduction in the number of jumps and a worsening of the qualitative characteristics of the semen produced . Also, the cryopreservation process influences the quality of the semen causing premature sperm capacitation hence, reducing its longevity in the female reproductive tract. At present, for cryopreservation of semen, standard protocols are used equally for all species, not considering the significant interspecific differences that exist. The aim of the research was, therefore, to evaluate the effect of season on buffalo frozen-thawed semen and to improve its quality by analyzing the effects of the addition of antioxidants such as resveratrol at various concentrations (0.5, 1, 10 and 50 uM), carnitine (2.5 and 7.5 mM ) and sterols, such as cholesterol (1.5 and 3 mg /ml), to the standard freezing extenders. The addition of antioxidants and / or sterol has stabilized the sperm membrane, reducing the state of capacitation, improving motility and consequently semen quality. The effect of the photoperiod was evident with a better semen quality during autumn. Based on these results was also evaluated fertilizing capacity both in vivo (resveratrol and cholesterol) and in vitro (resveratrol and carnitine)selecting concentrations that showed a more marked effect on the stabilization of the sperm membrane. The results obtained suggest that the premature capacitation after cryopreservation of spermatozoa is reduced during the favorable season and that the addition of antioxidants and / or sterols has a positive effect on buffalo semen quality and consequently, on in vitro and in vivo fertility.

The current knowledge on metabolism and glucose utilisation of preimplantation bovine and ovine embryos suggest the reduction of glucose concentration during early culture. On the contrary, it has been demonstrated that glucose is absolutely required for in vitro culture of buffalo embryos, as indicated by the poor efficiency recorded in the absence of this substrate during early embryonic development (Monaco et al. 2006 Reprod. Domest. Anim. 41, 332). However, complete removal of glucose from culture medium throughout pre-elongation development is unlikely to benefit the embryo because glucose plays other roles including ribose and NADPH production through the pentose-phosphate pathway. Therefore, the aim of this study was to investigate the effect of reducing glucose concentration up to 0.15 mM (1/10 compared to the standard concentration in SOF) on embryo development in buffalo. In order to evaluate the role of this substrate during development, glucose was reduced at different stages of embryo culture. Cumulus???oocyte complexes (n = 573, over 4 replicates), recovered from slaughtered animals, were matured and fertilized in vitro according to our standard procedures (Gasparrini et al. 2006, Theriogenology, 65, 275???287). On day 1 (Day 0 = IVF), zygotes were cultured in SOF with group A) 1.5 mM glucose (standard concentration in SOF) throughout culture (control); group B) 1.5 mM glucose for early culture (Day 1 to Day 4) and 0.15 mM glucose for late culture (Day 4 to Day 7); group C) 0.15 mM glucose throughout culture; and group D) 0.15 mM glucose for early culture and 1.5 mM glucose for subsequent culture. In vitro culture was carried out at 38.5°C under 5% CO2, 7% O2, and 88% N2. Cleavage rate was evaluated on Day 4, and blastocyst yield, in relation to cleaved embryos, was recorded on Day 7. Differences among groups in blastocyst rate were analysed by chi-square test. The reduction of glucose concentration did not affect cleavage rate (73.7 v. 65.1%, respectively, for Groups A-B and C-D). Nevertheless, blastocyst rates significantly decreased when glucose was reduced throughout culture (Group C: 10.1%; P < 0.01) and to a limited degree during early culture (Group D: 17.2%; P < 0.05) compared with the control (Group A: 38.3%). On the contrary, a decreased glucose concentration during late culture did not reduce embryo development (Group B: 35.18%). This finding indicates that energy requirements of buffalo embryos during IVC are different from those of sheep and cattle, which show a significant rise in glucose uptake just around compaction, i.e. during late culture (Thompson et al. 1991 Reprod. Fertil. Dev. 3, 571???576; Thompson et al. 1996 J. Reprod. Fertil. 106, 299???306). In conclusion, in buffalo, unlike sheep and cattle, glucose is more critical for early embryo development than for post-compaction development, suggesting the importance of developing other strategies for optimizing in vitro embryo production efficiency.

Most media employed for producing bovine embryos in vitro include glucose as an energy source despite putative toxic effects. The aim of this work was to evaluate whether replacing glucose with myo-inositol and citrate during IVC improves in vitro embryo development and resistance to cryopreservation. Abattoir-derived oocytes were matured and fertilized in vitro using standard procedures. After 20–22 h of gametes co-incubation, zygotes were denuded and cultured in SOF containing either 1.5 mM glucose or 2.77 mM myo-inositol and 0.34 mM citrate for 7 days. Embryos were first incubated in 7.5 % EG and 7.5 % dimethyl sulfoxide (DMSO) for 3 min, then transferred into 16.5 % EG and 16.5 % DMSO and 0.5 M sucrose for 25 sec before being loaded into the cryotop. Warming was carried out by immerging the cryotop into a 0.25 M sucrose solution and by transferring the embryos into a 0.15 M sucrose for 5 min. Vitrified-warmed embryos were then cultured in vitro for further 24 hr, after which the embryo survival rate was recorded. Data were analyzed by Chi-Square test. The results of this study showed that myo-inositol-citrate increased blastocyst yield (37.4 vs 29.5 %, respectively; P

Most systems for producing mammalian embryos in vitro use glucose as an energy source despite putative toxic effects. It is known that female embryos are more sensitive to negative effects of glucose during IVC. The aim of this work was to evaluate whether replacing glucose with myo-inositol and citrate during IVC affects sex ratio. Abattoir-derived oocytes were matured and fertilized in vitro using standard procedures. After 20–22 h of gametes co-incubation, zygotes were denuded and cultured in SOF containing either 1.5 mM glucose or 2.77 mM myo-inositol and 0.34 mM citrate, for 7 days. The percentages of blastocysts were recorded and the embryos (on average 122 per group) were sexed by PCR as previously described (Alomar, 2008, Anim. Reprod. Sci. 107 48-61.). Differences in blastocyst rates and in the percentages of female embryos between groups were analyzed by Chi-Square test. The results of this study showed that myo-inositol-citrate increased both blastocyst yield (37.4 vs 29.5 %, respectively; P

The objectives of this work were to evaluate whether the sperm penetration speed is correlated to the in vitro fertility and whether adapting the gamete co-incubation length to the kinetics of the bull improves in vitro fertility and affects the sex ratio. In vitro matured oocytes were co-incubated with spermatozoa from four different bulls (A-D). At various post-insemination (p.i.) times (4, 8, 12, 16 and 20 h), samples of oocytes were fixed and stained with DAPI for nuclei examination, while the remaining ones were transferred into culture to evaluate embryo development. The blastocysts produced were sexed by PCR. Two bulls (A and B) had faster kinetics than the others (C and D), as shown by the higher penetration rates recorded at 4 h p.i. (43%, 30%, 11% and 6%, respectively for bulls A, B, C and D; p

Removal of cumulus cells is necessary for several technologies such as vitrification, intracytoplasmic sperm injection, and nuclear transfer. However, it is known that the presence of cumulus cells during IVF of buffalo oocytes is fundamental for fertilization and embryo development (Gasparrini et al. 2007 Anim. Reprod. Sci. 98, 335–342; Nandi et al. 1998 Theriogenology 50, 1251–1262). The aim of this work was to evaluate whether co-culture with intact bovine cumulus–oocyte complexes (COC) during IVF would restore the developmental competence of denuded buffalo oocytes. Due to the scarce availability of buffalo ovaries, the somatic support was provided by bovine cumulus cells. Abattoir-derived COC were matured in vitro according to our standard procedures (Gasparrini et al. 2006, Theriogenology, 65, 275–287) and randomly distributed in 3 fertilization groups: 1) a control group of COC (n = 122), 2) a negative control of denuded oocytes (DO; n = 119), and 3) DO co-cultured with in vitro matured bovine COC (DO+COC; n = 103) in a 1:1 ratio (3 bovine COC + 3 denuded buffalo oocytes/50 μL drop). Fertilization was carried out with frozen–thawed spermatozoa from a tested bull in TALP medium supplemented by 0.2 mM penicillamine, 0.1 mM hypotaurine, and 0.01 mM heparin at 38.5°C under a controlled gas atmosphere of 5% CO2 in humidified air. After fertilization the zygotes were cultured in SOF medium including essential and nonessential amino acids and 8 mg mL–1 BSA, at 38.5°C under humidified 5% CO2, 7% O2, and 88% N2, up to the blastocyst stage. On Day 5 and on Day 7 (Day 0 = IVF) cleavage and blastocyst rates were respectively recorded. Data were analysed by chi-square test. As expected, cleavage and blastocyst rates were lower (P < 0.01) in DO (36.1 and 9.2%, respectively) compared with the control (67.2 and 27.1%, respectively). However, co-culture during IVF (DO+COC) significantly increased (P < 0.01) both parameters compared with DO, giving cleavage (70.9%) and blastocyst (27.2%) rates similar to the control. The results of this study demonstrated that co-culture with bovine intact COC during IVF of buffalo denuded oocytes completely restores their fertilizing capability and blastocyst developmental competence. We conclude that this may be a suitable strategy for preserving the developmental competence of oocytes devolved to technologies, such as oocyte vitrification, that require cumulus removal.

Most systems for producing mammalian embryos in vitro use glucose as an energy source in the media despite putative toxic effects (Schini and Bavister 1988 Biol. Reprod. 39, 1183???1192; Takahashi and First 1992 Theriogenology 37, 963???978). Currently there is a tendency to identify other suitable energy sources in an attempt to replace glucose from culture media. Glyceraldehyde-3-phosphate (G3P), a glucose-derived high-energy compound, is the end product of the energy-consuming phase of glycolysis that enters the pay-off phase of the pathway characterised by a net gain of energy. The aim of this study was to determine whether G3P is a valid energy source for supporting in vitro embryo development in cattle. Abattoir-derived oocytes (n = 832, over 4 replicates) were matured in vitro in TCM-199 with 15% bovine serum (BS), 0.5 ??g mL???1 FSH, 5 ??g mL???1 LH, 0.8 mM L-glutamine, and 50 mg mL???1 gentamicin. Mature COC were fertilized in Tyrode???s modified medium, with 30 mg mL???1 heparin, 30 mM penicillamine, 15 mM hypotaurine, 0.15 mM epinephrine, and 1% BS. Both IVM and IVF were carried out at 39°C and 5% CO2 in air. After 20 to 22 h of gamete co-incubation, presumptive zygotes were denuded and cultured in SOF containing either 1.5 mM glucose (control group) or G3P at 3 different concentrations (0.125, 0.5, and 1.5 mM). It is worth specifying that in the 3 G3P-supplemented groups small amounts of glucose were left (0.15 mM) because it is known that a complete removal would affect embryo development by interfering with ribose synthesis through the pentose???phosphate pathway. In vitro culture was carried out at 39°C under humidified air with 5% CO2, 7% O2, and 88% N2 in air for 7 days, when the percentages of tight morulae-blastocysts (TMBL) and superior quality blastocysts (BL) were recorded. Differences in embryo yields among groups were analysed by chi-square test. Supplementation of IVC medium with 1.5 mM G3P reduced (P < 0.01) TMBL (5.0%) and BL (5.0%) rates compared with all other groups, indicating a toxic effect. However, when G3P was added at lower concentrations, no differences in TMBL (37.3 and 26.1, respectively, with 0.125 and 0.5 mM G3P) and in BL rates (35.3 and 25.5%, respectively, with 0.125 and 0.5 mM G3P) were observed compared with the control (32.7% TMBL and 31.4% BL, respectively). Within G3P-treated groups, the higher embryo yields were recorded with 0.125 mM compared with 0.5 mM (P < 0.05) and 1.5 mM (P < 0.01). Interestingly, embryos produced with G3P at the lower concentrations (0.125 and 0.5 mM) seemed to show a faster development compared with the control. In conclusion, these results demonstrated that G3P is a valid energy source for bovine preimplantation embryos and, hence, that G3P supplementation of IVC medium may be a suitable option for reducing glucose concentration in the media. However, further studies are needed to investigate lower concentrations of G3P and to better evaluate embryo viability.

The aim of this research was to detect possible relations between enzymes activity and sperm function in order to evaluate the potential use of enzymaic tests as markers of sperm quality. Results showed that all enzymes tested are involved in sperm metabolism and their activity is related to sperm quality. Correlation was found in seminal plasma and sperm cells from both fresh and frozen semen thus showing the potential use of enzymati tests to evaluate in vitro sperm fertility

The aim of the present study was to compare two synchronization protocols for timed artificial insemination (TAI) in acyclic pluriparous buffalo cows during the non breeding season. Two experiments were conducted to evaluate the ovarian follicular response and pregnancy rate. The cyclic status was evaluated by two transrectal ultrasonography performed at Day -11 and Day 0. Buffaloes that in both investigations did not show the presence of a corpus luteum (CL) were classified as acyclic. Acyclic pluriparous buffaloes (n=34) were randomly assigned to Group 1 (G1) and Group 2 (G2), homogeneous for Days in Milk (81±27 vs 83±13, respectively in G1 and G2). In G1 (n=17) buffaloes received 12μg of buserelin acetate i.m. (GnRH) on the first day of the synchronization protocol (Day 0), 0.524 mg of cloprostenol (Pgf2α) on Day 7 and 12μg of buserelin acetate i.m. on day 9 (Ovsynch-TAI). In G2 (n=17) buffaloes received a progesterone-releasing intravaginal device containing 1.55 g of progesterone (P4) and 12μg of buserelin acetate i.m. (GnRH) on Day 0. On Day 8 the P4 device was removed and 0.524 mg of cloprostenol (PGF2α) + 500UI of PMSG i.m. were administered. Finally, 12μg of buserelin acetate i.m. were given on Day 10. Ten animals (5/group) underwent transrectal ultrasonography of the ovaries daily, from Day 0 to Day 11 in G1 and from Day 0 to Day 12 in G2, to determine the presence and diameter of the follicles, the dominant follicle (DF) diameter and the ovulation rate. Subsequently, fixed TAI was performed 20 hours after the last GnRH in all buffaloes. Ultrasonography was carried out 25 and 45 days after TAI to evaluate pregnancy rate and the incidence of late embryonic mortality (LEM). No differences were observed between G1 and G2 in the following parameters: DF diameter (mm) on Day 0 (9.5±1.9 vs 8.8±4.3), DF diameter (mm) on Day of Pgf2α administration (11.5±2.3 vs 10.6±1.6), DF growth rate (mm) between PGF2α and last GnRH administration (1.6±0.3 vs 1.8±1.7), DF maximum diameter (13.2±1.9 vs 13.0±2.3 mm) and ovulation rate (80% vs 80%). However, in G2 pregnancy rate increased at 25 (29.4% vs 58.8%, respectively in G1and G2; P= 0.08) and 45 (23.5% vs 58.8%, respectively in G1and G2; P

Antioxidant supplementation has been proposed as a new strategy to improve the long-term preservation of semen. The aim of this study was to evaluate the effect of Maca supplementation of semen extender on quality-related canine semen parameters during cooling. Ejaculates from nine dogs were cooled for 7 days in the absence (control group) or in the presence of 10, 20 and 50 μL/mL of an aqueous extract of Maca. Sperm were evaluated for sperm viability, motility, DNA fragmentation and lipid peroxidation after 3 h, 24 h, 4 days and 7 days of storage. The addition of 10 μL/mL of Maca preserved sperm DNA and plasma membrane integrity at 3 h and increased sperm curvilinear velocity after 24 h. Treatment with 20 and 50 μL/mL of Maca increased the percentage of hyperactivated sperm after 3 h. Moreover, semen treated with 20 μL/mL of Maca decreased lipid peroxidation at 24 h. A significant reduction of sperm DNA and plasma membrane integrity as well as of kinetics parameters between 3 and 24 h of refrigerated storage with the higher concentration tested was observed. Although Maca was not able to protect canine semen with extended refrigeration storage time, it increased hyperactivation and preserved DNA integrity in short-term storage.

In buffalo (Bubalus bubalis) reproductive seasonality, causing cycles of milk production, is one of the major factors affecting farming profitability. Follicular fluid (FF) contains extracellular vesicles (EVs) playing an important role in modulating oocyte developmental competence and carrying microRNAs (miRNAs) essential for in vitro fertilization outcomes. The aim of this work was to characterize the FF-EVs-miRNA cargo of antral (An) and preovulatory (pO) follicles collected in the breeding (BS) and non-breeding (NBS) seasons, to unravel the molecular causes of the reduced oocyte competence recorded in buffalo during the NBS. In total, 1335 miRNAs (538 known Bos taurus miRNAs, 324 homologous to known miRNAs from other species and 473 new candidate miRNAs) were found. We identified 413 differentially expressed miRNAs (DE-miRNAs) (FDR

The aim of this study was to evaluate the treatment of bovine semen with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK), before or after freezing on semen quality. After the initial assessment, sperm from 4 bulls were pooled (Experiment 1) and cryopreserved in BioXcell containing 0, 20 and 100 μM Z-VAD-FMK. After thawing semen viability, motility, membrane integrity, as well as DNA fragmentation and ΔΨm were evaluated. In Experiment 2, bovine frozen/thawed sperm were incubated for 1 hr with 0, 20 and 100 µM Z-VAD-FMK before assessing the semen quality. The treatment with Z -VAD-FMK before cryopreservation improved post-thawing sperm motility compared to the control group (p < .05), while no differences were recorded in sperm viability and membrane integrity among groups (on average 86.8 ± 1.5 and 69.1 ± 1.4, respectively). Interestingly, at the highest concentration, DNA fragmentation decreased (p < .05), while the percentage of spermatozoa with high ΔΨm increased (p < .05). The results of Experiment 2 showed that 1-hr treatment with Z-VAD-FMK did not affect sperm motility and viability (on average 63.4 ± 5.8 and 83.7.1 ± 1.2, respectively). However, Z-VAD-FMK improved sperm membrane integrity (p < .05) and at the highest concentration tested decreased the proportion of sperm showing DNA fragmentation (p < .05). No differences were recorded in the percentage of spermatozoa with high ΔΨm (on average 57.0 ± 11.4). In conclusion, the treatment with 100 µM of the caspase inhibitor Z-VAD-FMK before freezing increased bovine sperm mass motility and ΔΨm, while decreasing sperm DNA fragmentation. Treatment of semen after thawing with 100 µM Z-VAD-FMK improved sperm membrane integrity and reduced DNA fragmentation.

The aim of this study was to evaluate the effect of resveratrol supplementation of semen extender on fertility parameters of frozen-thawed buffalo sperm. After the initial semen assessment, buffalo semen was cryopreserved in BioXcell containing 0 (control group), 0.5, 1, 10, and 50-μM resveratrol. After thawing, viability, motility, and capacitation status (assessed by localization of phosphotyrosine-containing proteins) were evaluated. Based on the results of the dose-response trial, the concentration of 50 μM was selected for further assessments, such as membrane integrity, total antioxidant capacity, reactive oxygen species, and lipid peroxidation (LPO) levels. Moreover, in vitro fertilizing ability by heterologous IVF and in vivo fertility were assessed. No differences among groups were recorded in sperm motility and viability (on average 52.3 ± 2.1% and 76.6 ± 1.3%, respectively). However, data showed a resveratrol dose-dependent effect on sperm capacitation status, with a significant reduction of the cryopreservation-induced capacitation with the higher concentrations tested. In particular, both 10- and 50-μM resveratrol increased (P

The aim of this work was to evaluate whether the residual amount of γ-glutamyl-transferase (GGT) and alkaline phosphatase (ALP) in bovine sperm after freezing/thawing is correlated with fertility parameters, including blastocyst rates after in vitro fertilisation (IVF). The enzyme activities were determined in both spermatozoa and supernatant after centrifugation. While ALP was only correlated with sperm viability, GGT activity was correlated with sperm motility (rs = .4; p

The aim of this work was to evaluate the effect of relaxin on fertility parameters of buffalo frozen/thawed sperm. Sperm were incubated in the absence of capacitating agents (negative control), with a known capacitating agent such as heparin (positive control) and with 50 and 100 ng/ml relaxin for 2 and 4 h. Sperm viability, motility, capacitation and the effect of relaxin on the fertilizing ability after heterologous IVF were evaluated. Although viability was not affected, relaxin increased (p < 0.05) sperm motility compared to the negative and positive controls both after 2 h (60.0 ± 2.0, 60.0 ± 3.1, 68.3 ± 1.7 and 69.4 ± 2.7, respectively, in negative control, positive control, 50 and 100 ng/ml relaxin) and 4 h (55.0 ± 2.5, 53.3 ± 3.0, 62.2 ± 3.0 and 65.0 ± 3.2, respectively, in negative control, positive control, 50 and 100 ng/ml relaxin) incubation. When sperm were incubated with both 100 ng/ml relaxin and heparin, a decrease (p < 0.01) of pattern A, that is low capacitation level, was observed compared to the negative control both after 2 h (54.4, 34.3 and 36.4%, respectively, in negative control, positive control and 100 ng/ml relaxin) and 4 h (51.9, 35.0 and 34.3%, respectively, in negative control, positive control and 100 ng/ml relaxin). Moreover, an increase (p < 0.01) of pattern EA, that is high capacitation level, was recorded with 100 ng/ml relaxin and heparin compared to the negative control both after 2 h (44.1, 59.3 and 57.7%, respectively, in negative control, positive control and 100 ng/ml relaxin) and after 4 h (43.0, 54.4 and 56.0%, respectively, in negative control, positive control and 100 ng/ml relaxin). Finally, relaxin increased (p < 0.01) cleavage rate compared to the negative control (57.1 ± 4.4, 72.5 ± 6.0, 71.4 ± 5.5 and 73.6 ± 2.9, respectively, in negative control, positive control, 50 and 100 ng/ml relaxin). In conclusion, relaxin has a beneficial effect on motility, capacitation and fertilizing ability of frozen-thawed buffalo sperm.

Contents Aim of this study was to test the reliability of Trypan blue/Giemsa staining to evaluate sperm membrane integrity, acrosomal intactness and morphology in stallion to verify whether it could be applied in vitro as useful tool for sperm fertilizing ability. Fertility data on inseminated mares were collected to evaluate the relationship of sperm quality to pregnancy rates. Forty-one ejaculates were collected from 3 stallions of Salernitano Horse Breed and evaluated for gross appearance, volume, visual motility and membrane integrity with Trypan blue/Giemsa staining and thirty-five mares were inseminated during the breeding season from April to July. Differences among stallions were found in volume, sperm concentration (p

The aim of this study was to investigate the effect of cholesterol-loaded cyclodextrins (CLC) on motility, viability, capacitation status, and in vivo fertility of buffalo frozen-thawed sperm. After the initial semen assessment, buffalo sperm were diluted in BULLXcell extender containing 0- (control), 1.5-, and 3-mg/mL CLC and cryopreserved. At thawing, sperm motility was evaluated by phase contrast microscopy, and viability-capacitation status was assessed by Hoechst 33258-chlortetracycline (CTC) assay. Capacitation status was also evaluated by an indirect immunofluorescence assay to localize phosphotyrosine-containing proteins. Moreover, buffaloes were artificial inseminated to assess the in vivo-fertilizing potential of CLC-treated semen. No differences among control, 1.5-, and 3-mg/mL CLC-treated groups were recorded in both sperm motility (66.5 ± 5.6, 68.8 ± 4.8, and 68.8 ± 4.8, respectively) and viability (86.5 ± 1.9, 87.6 ± 1.5, 88.4 ± 2.3, respectively). However, the extender supplementation with CLC significantly reduced sperm cryocapacitation. Indeed, CLC treatment decreased (P < 0.01) the proportion of sperm showing the CTC pattern B (capacitated sperm) compared with the control (69.6 ± 3.4, 37.8 ± 1.5, and 51.3 ± 4.7, respectively, with 0, 1.5-, and 3-mg/mL CLC; P < 0.01). Furthermore, the percentage of sperm displaying tyrosine-phosphorylated pattern EA (i.e. high capacitation level) was reduced (P < 0.01) in both CLC-treated groups (10.8 ± 3.3 and 5.6 ± 1.6, respectively, with 1.5- and 3-mg/mL CLC) compared with the control (37.3 ± 6.9), reaching values similar to those recorded in fresh semen (11.0 ± 3.5). In addition, treating sperm with 3-mg/mL CLC increased (P < 0.01) the percentage of nonfluorescent (pattern NF), i.e., non-capacitated sperm (41.8 ± 3.6) compared with fresh semen (11.0 ± 6.9). No differences were recorded in pregnancy rates at 60 days post-artificial insemination among control, 1.5- and 3-mg/mL CLC groups (59.7%, 65.6%, and 56.9%, respectively). In conclusion, CLC treatment of buffalo sperm strongly decreases sperm cryocapacitation damages, without affecting the in vivo fertilizing capability.

The aim of this study was to evaluate the effect of osteopontin (OPN), an ubiquitous acid glycoprotein, on in vitro sperm capacitation and on in vitro embryo production (IVEP) efficiency in buffalo. In experiment 1, after swim-up separation the sperm were incubated in Tyrode albumin lactate pyruvate medium in the absence of capacitating agents (control), with the standard concentration of heparin (0.01 mM) and three different concentrations of OPN (0.1, 1, and 10 mcg/mL), both in the presence and absence of heparin, for 2 and 4 hours. Capacitation was assessed indirectly by estimating the percentage of acrosome-reacted sperm after incubation with lysophosphatidylcholine. In order to determine the effect of OPN, in the presence of heparin, on fertilization (Experiment 2) and in vitro embryo development (experiment 3), in vitro-matured buffalo oocytes were fertilized in the presence of 0, 0.1, 1, and 10 mcg/mL of OPN. After IVF, the presumptive zygotes were dezonated, fixed, stained, and then evaluated microscopically. At Days 5 and 7 of culture, the cleavage and blastocyst rates were evaluated, respectively. Two hours of treatment with OPN at the two higher concentrations (1 and 10 mcg/mL) promoted in vitro capacitation of buffalo sperm (experiment 1). A synergic action of OPN with heparin was also done for all OPN concentrations tested. At 4 hours incubation, all treatments, including heparin (20.4%), improved (P < 0.01) capacitation compared with the control (16.2%). Interestingly, the best results were reported in all groups treated with OPN + heparin (40.8%, 38.6%, and 33.8%, respectively; P < 0.01). The addition of OPN to the IVF medium had a positive influence on total penetration, synchronous pronuclei formation (experiment 2), and IVEP efficiency (experiment 3). In particular, the two lower concentrations of OPN (0.1 and 1 mcg/mL), compared with the control, gave higher synchronous pronuclei formation (73.5%, 75.0%, and 46.5%, respectively; P < 0.01) and cleavage rates (70.3%, 71.6%, and 59.3%, respectively; P < 0.01). Interestingly, the treatments also improved blastocyst yields (29.3%, 30.3%, and 19.4%, respectively; P < 0.01). In conclusion, these results indicate that adding OPN to the IVF system improves IVEP efficiency by enhancing in vitro sperm capacitation and blastocyst yields in buffalo.