Search

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.

The peculiar reproductive characteristics of the species limit the utilization of artificial insemination (AI) in buffalo. For this reason, in the last years a great attention has been focused on estrus synchronization methods based on fixed timed artificial insemination (FTAI). Among these, the Ovsynch-TAI program has been successfully applied in buffalo. The aim of this study was to assess the response to the first GnRH, prostaglandin and second GnRH in buffaloes synchronized by Ovsynch and subsequent pregnancy outcome. The trial was carried out on 59 buffaloes (138.7±86 days in milk) bred in a commercial farm in the South of Italy. Ten days before the start of the study all the animals underwent clinical and ultrasound examination, to confirm the absence of any gross abnormalities of the genital tract. Synchronization of the estrous cycle was performed by Ovsynch-TAI program which involved the injection of a GnRH agonist on Day 0 (12 μg of buserelin acetate), prostaglandin on Day 7 (5 mg of Dinoprost), and a GnRH agonist again on Day 9 (12 μg). The AI was carried out 16 h after the second GnRH injection, using frozen-thawed semen from bulls of proven fertility. The ultrasound examinations of the ovaries were performed by using a portable Sonoace Pico equipped with a 10 MHz linear transducer adapted for transrectal examination in large domestic animals on Day 0, 1, 7, 9, 10 and 11. All visible antral follicles were recorded and classified into three categories, according to their size: small (diameter < 0.5 cm), medium (diameter between 0.5 and 1 cm) and large (diameter > 1cm). The dimensions of the dominant follicle and the corpus luteum (CL) were further registered. In order to evaluate the characteristics of the CLs, the colour-doppler mode was activated in order to display signals for blood flow in vessels. Pregnancy diagnosis was carried out on 35 days post-AI and the statistical analysis was performed by ANOVA and chi-square test. It was observed that 37 buffaloes (62.7%) ovulated after the first GnRH treatment and showed a functional corpus luteum on day 7. Interestingly, animals that ovulated after the first GnRH injection showed a lower (P=0.08) CL area (22.1±10.1 vs. 28.6±10.3 mm, respectively) and a larger follicle (10.5±4.3 vs. 8.5±3.0, respectively) on day 0. A total of 54 buffaloes (91.5%) showed a functional CL on day 7, although luteolysis occurred only in 50 subjects (84.7%). All the buffaloes (37/37) that ovulated after the first GnRH injection underwent luteolysis. On the contrary, CLs responsive to prostaglandins were recorded only in 59% (n=13) of the animals that did not ovulate after the first GnRH (P

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.

The aim of this study was to evaluate water balance and nitrogen excretion in Mediterranean buffaloes. Sixteen subjects were divided into 4 groups (n=4/group), according to their days in milk (DIM): Group L50 (DIM=50±8); Group L125 (DIM=125±55); Group L225 (DIM=225±26); Group NL (non-lactating). The study comprised a 14-day adaptation to diets and 14-day data collection during which feed intake and milk production were recorded daily. Measurements for individual buffaloes were made for water intake, diet, orts, milk, faeces and urine during the final 3 days. The analyses were performed according to OAOC. Lactating buffaloes had greater (P

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

In cattle, in vitro-produced (IVP) bovine embryos have been around in large numbers for over two decades. It is probably fair to say that their main use has been in research, as a means of understanding what happens during normal in vivo embryo development in cattle, as stepping stones to other technologies, such as nuclear transfer, and as a tool for examining what goes wrong in development following such procedures, and as models from which to extrapolate findings to other species. The high incidence of developmental failure in the in vitro embryo production system has been also attributed to suboptimal culture conditions that induce oxidative stress. Oxidative stress has been implicated in many different types of cell injuries, including membrane lipids peroxidation, oxidation of amino acids and nucleic acids, apoptosis and necrosis which may subsequently decrease the viability of IVP embryos. To overcome this, exogenous antioxidant compounds have been frequently used to increase antioxidant capacity of embryos via increasing intracellular levels of reactive oxygen species (ROS) scavengers. Development of accurate laboratory methods to assess embryo quality will improve the efficiency of embryo production from in-vitro culture systems. Currently, the techniques of TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling (TUNEL) for the detection of apoptosis, and differential staining for determining the ratio of inner cell mass (ICM) to trophectoderm (TE) cells, are used separately to assess embryo quality in a range of different species. Furthermore, the ability to cryopreserve embryos, without critical loss of viability, has a profound effect on the success of assisted reproductive techniques. The survival of cryopreserved in vitro produced (IVP) embryos, as measured either by post-warming survival in culture or by established pregnancies after embryo transfer, has lagged behind that of in vivo-derived embryos. Even if the background of this difference is not completely understood, it is known that the culture medium has dramatic effect on developmental competence and cryo-withstand of the resulting embryos. The aim of this research was to evaluate whether supplementation of bovine culture medium with different natural antioxidant compounds such as Resveratrol, L-Ergothioneine and Crocetin, found in several vegetal sources, improves in vitro blastocyst development and quality, assessed as resistance to cryopreservation, total cells number, ratio of ICM:TE cells and apoptosis index. Abattoir-derived oocytes were matured and fertilized in vitro according to our standard procedure. Twenty h after IVF, presumptive zygotes were cultured in SOF medium, supplemented with different concentrations of each antioxidant compound such as resveratrol (0, 0.25, 0.5 and 1 µM), L-ergothioneine (0, 0.05, 0.1 and 0.5 mM) and Crocetin (0, 1, 2.5 and 5 µM) 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 were vitrified by cryotop method in 16.5% ethylene glycol, 16.5% dimethyl sulfoxide (DMSO) and 0.5 M sucrose. Finally, blastocysts produced on day 8 in the absence (control) and presence of 0.5 µM resveratrol, 0.1mM L-ergothioneine and 1 µM Crocetin were used for TUNEL and differential staining to evaluate respectively the apoptotic rate and the allocation of cells into ICM and TE lineages. The results showed that: Resveratrol supplementation did not increase blastocyst yields, but 0.5 µM resveratrol improved the cryotolerance of IVP embryos compared to the control, as indicated by higher development rates and hatching rates recorded after 48 h post-warming culture. Blastocysts produced in the absence (control) and presence of 0.5 µM resveratrol had a similar number of ICM, TE and total cells, as well a similar ratio of ICM:total cells. L-ergothioneine supplementation did not increase blastocyst yields, but 0.1 mM L-ergothioneine group showed a better cryotolerance of IVP embryos compared to the control group, as indicated by higher hatching rates recorded after 48 h post-warming culture. In addition, blastocysts produced in the presence of 0.1 mM L-ergothioneine have proved a higher embryo quality compared to the control, as indicated by lower percentage of apoptotic rate and higher percentage of blastocysts with the most physiological ICM:total cells ratio. In contrast to previous antioxidant compounds, Crocetin supplementation of 1 µM increase blastocyst yields in term of higher total embryo output, superior quality blastocysts and fast developing embryos percentages. In addition, this crocetin concentration improved the cryotolerance of IVP embryos compared to the control, as indicated by higher hatching rates recorded after 48 h post-warming culture. Blastocysts produced in the absence (control) and presence of 1 µM crocetin had a similar number of ICM, TE and total cells, as well a similar ratio of ICM:total cells. Furthermore, blastocysts produced in IVC medium enriched with 1 µM crocetin have proved a higher embryo quality compared to the control, showing a lower percentage of apoptotic rate.

The present study aimed to assess the applicability of luteal blood flow data acquired through the use of color Doppler ultrasonography and a post-processing analysis tool (ImageJ) for predicting pregnancy in buffaloes (Bubalus bubalis). The experiment was carried out on 59 multiparous Italian Mediterranean buffaloes that underwent synchronization of estrus and fixed-time artificial insemination (TAI). Corpus luteum features (size: CLS and blood flow: BFA) were taken from Day 5 to 10 after TAI and retrospectively measured with ImageJ. In the same period, blood samples were taken to assess progesterone (P4) concentrations. Pregnancy diagnosis was carried out on Day 45 by ultrasound and confirmed on Day 70 post-TAI. Differences in CLS, BFA, and P4 concentrations from Day 5 to 10 after TAI measured between groups were analyzed by ANOVA repeated measures as were differences within each day of measuring. Buffaloes that established a pregnancy (n = 29; 55%) had larger CLS (2.2 ± 0.1 vs. 1.9 ± 0.1 cm2; P < 0.01), higher BFA (0.6 ± 0.0 vs. 0.4 ± 0.0 cm2; P < 0.01), and higher P4 blood level (1.8 ± 0.1 vs. 1.4 ± 0.1; P < 0.01) during Day 5–10 as compared to not-pregnant buffaloes (n = 22). Throughout the entire period, the first feature that changed between groups was P4 blood concentration at Day 7 (1.7 ± 0.1 vs. 1.2 ± 0.1; P < 0.05) followed by BFA at Day 8 (0.6 ± 0.0 vs. 0.5 ± 0.0; P < 0.05), respectively, in pregnant and not-pregnant animals. The ROC analyses indicated that P4 was able to predict pregnancy since Day 5 (P < 0.05) although a more reliable result could be obtained from Day 8 (P < 0.01). At Day 10, it was possible to set a cutoff value for every parameter taken into account. The logistic regression analysis showed that pregnancy was positively influenced by P4 concentration (odds ratio 534.127; P < 0.01) and BFA (odds ratio 744.893; P < 0.01). In conclusion, the use of color Doppler ultrasonography, together with ImageJ, identified different patterns of BFA between pregnant and not-pregnant buffaloes starting from Day 8 post-TAI.

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 factors affecting ovum capture in superovulated buffaloes, by comparing the morphological features of pre-ovulatory follicles and oocytes, the intrafollicular and plasmatic steroid profile, as well as the expression of genes involved in cumulus expansion and steroid cascade in granulosa cells (GCs) and that of genes involved in contraction-relaxation of the oviduct between superovulated and synchronized buffaloes. Italian Mediterranean Buffalo cows were either synchronized by Ovsynch (n = 25) and superovulated (n = 10) with conventional FSH protocol and sacrificed 18 h after last GnRH. Antral follicular count, recovery rate and oocyte quality were recorded, and plasma and follicular fluid were collected for steroid profile determination. In addition, in 10 animals (5/group), GCs were collected to analyse the mRNA expression of gonadotropin receptors (LHR and FSHR) and genes involved in steroid synthesis, as the cytochrome P450 family 19 (CYP19A1) and the steroidogenic acute regulatory protein (STAR). Moreover, oviducts were collected to evaluate the mRNA expression of estrogen receptor 1 (ER1) and the progesterone receptor (PGR), the vascular endothelial growth factor (VEGF) and the VEGF receptors, i.e. the kinase insert domain receptor (FLK1) and the fms related tyrosine kinase 1 (FLT1). No differences were recorded in steroids plasma concentration between synchronized and superovulated animals whereas intrafollicular E2 and P4 concentrations decreased in superovulated group (63.2 ± 10.6 vs 30.3 ± 5.9 ng/mL of E2 and 130.1 ± 19.8 vs 71.6 ± 8.5 ng/mL of P4, respectively in synchronized and superovulated animals; P < 0.05). Interestingly, both the recovery rate (85.7% vs 56.6%, respectively in synchronized and in superovulated animals; P < 0.05) and the percentage of oocytes exhibiting proper cumulus expansion (75% vs 28.1%, respectively in synchronized and in superovulated animals; P < 0.01) decreased in superovulated animals. In addition, the expression of FSHR and CYP19A1 increased while the expression of STAR in GCs decreased (P < 0.05). Finally, in superovulated buffaloes a decreased expression of PGR, ER1, VEGF and its receptor FLK1 in the oviduct was observed. The results suggest that the exogenous FSH treatment impairs steroidogenesis, affecting both the oviduct and the ovarian function, accounting for the failure of ovum capture in superovulated buffaloes.

The aim of this work was to evaluate whether the treatment with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) during cryopreservation and post warming in vitro culture improves cryotolerance of bovine in vitro produced (IVP) embryos. Abattoir derived bovine oocytes were in vitro matured, fertilized and cultured according to standard procedure. On Day 7, embryo yields were assessed and blastocysts randomly divided in 2 groups: vitrification and post-warming culture in the absence (n = 184) or presence (n = 156) of 20 mu M Z-VAD-FMK. Resistance to cryopreservation was evaluated post-warming culture by assessing the survival rate and hatching rate. Differential staining combined with in situ terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL) technique was performed to evaluate total cells number, cell allocation into inner cell mass (ICM) and trophectoderm (TE) lineages, as well as the DNA fragmentation rate of vitrified blastocysts, while immunohystochemical staining was used to assess the level of cleaved-caspase 3. It was demonstrated that inhibition of caspase activity by Z-VAD-FMK increases embryo cryotolerance, as indicated by higher survival (76.1 vs 51.1%; P < 0.01) and hatching rates (26.5 vs 17.6%; P < 0.05) after 48 h of post-warming culture. Furthermore, Z-VAD-FMK decreased both the average number (4.7 +/- 03 vs 7.7 +/- 0.5; P < 0.01) and the percentage (3.4 +/- 0.2 vs 6.1 +/- 0.5; P < 0.01) of DNA fragmented cells in blastocysts compared to the control. No differences were recorded in the average number of ICM, TE and total cells between groups. The level of cleaved-caspase-3, the downstream effector of apoptosis, and its relative percentage on total area of blastocysts was reduced (P < 0.01) in the presence of Z-VAD-FMK both at thawing (1.29 +/- 0.17 vs 3.24 +/- 0.46) and after 48 h post-warming culture (1.46 +/- 0.17 vs 5.06 +/- 0.41). In conclusion, the addition of 20 mu M Z-VAD-FMK during vitrification/warming and post warming culture partially inhibits cryopreservation-induced apoptosis by reducing the level of active caspase 3, suggesting a potential use as an additive to ameliorate the efficiency of embryo cryopreservation in cattle, critical for a further diffusion of IVEP technology in the field. Further studies are though needed to evaluate the effect of Z-VAD-FMK on post-transfer embryo development before considering a commercial application.

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.

The aim of the study was to evaluate whether resveratrol supplementation of bovine culture medium improves in vitro blastocyst development, embryo cryotolerance and cell numbers. Abattoir-derived oocytes were matured and fertilized in vitro according to standard procedure. Twenty hours after IVF, zygotes were cultured in SOF medium, supplemented with 0 (control, n=439), 0.25μM (n=422), 0.5μM (n=447) and 1μM resveratrol (n=416). On Day 7 (IVF=Day 0) blastocysts were vitrified by cryotop in 16.5% ethylene glycol, 16.5% dimethyl sulfoxide and 0.5M sucrose. Development rate, i.e. the percentage of embryos resuming development to reach a more advanced stage, and hatching rate were evaluated after 24 and 48h culture. Blastocysts cultured with (0.5μM) and without resveratrol underwent differential staining to count inner cell mass (ICM) and trophectoderm (TE) cells. Resveratrol during culture did not increase blastocyst yields (57.1, 57.7, 59.2 and 46.6%, respectively in 0, 0.25, 0.5 and 1μM resveratrol). However, 0.5μM resveratrol improved embryo cryotolerance compared to the control, as indicated by higher development rates (67.3% vs 50.3%, respectively; P

The aim of this work was to assess the effect of supplementation of bovine culture medium with the natural antioxidant crocetin on in vitro blastocyst development and quality. This was evaluated as cryotolerance, apoptosis index, and total cells number and allocation. Abattoir-derived oocytes were matured and fertilized in vitro according to standard procedure. Twenty hours after IVF, presumptive zygotes were cultured in synthetic oviduct fluid medium, supplemented with 0, 1, 2.5, and 5 μM crocetin (experiment 1) at 39 °C under humidified air with 5% CO2, 7% O2, and 88% N2. On Day 7, embryo yields were assessed and the blastocysts were vitrified by Cryotop method in 16.5% ethylene glycol, 16.5% DMSO, and 0.5 M sucrose. Finally, blastocysts produced on Day 8 in the absence (control) and presence of 1 μM crocetin were used for terminal deoxynucleotidyl transferase–mediated dUTP nick end labelling and differential staining to evaluate, respectively, the apoptotic rate and the allocation of cells into inner cell mass (ICM) and trophectoderm (TE) lineages (experiment 2). Embryo development was higher in the 1 μM crocetin group compared to the control, both in terms of total embryo output (37.7 ± 4.2%, 52.9 ± 6.3%, 40.9 ± 7.6%, and 42.4 ± 8.7%, respectively, with 0, 1, 2.5, and 5 μM; P

The aim of this study was to evaluate whether supplementation of bovine culture medium with the natural antioxidant L-ergothioneine (LE), improves in vitro blastocyst development and quality, assessed as resistance to cryopreservation, total cells number, cellular differentiation, and apoptosis index. Abattoir-derived oocytes were matured and fertilized in vitro according to standard procedure. Twenty hours after IVF, presumptive zygotes were cultured in synthetic oviduct fluid with 0, 0.05 mM, 0.1 mM, 0.5 mM, and 1 mM of LE (experiment 1) at 39 °C under humidified air with 5% CO2, 7% O2, and 88% N2. On the basis of the results of this dose-response trial, the range of concentrations to test was reduced in experiment 2, in which presumptive zygotes were cultured with 0, 0.05 mM, and 0.1 mM of LE. On Day 7, embryo yields were assessed, and the blastocysts (BL) were vitrified by Cryotop method in 16.5% ethylene glycol, 16.5% DMSO and 0.5 M sucrose. Finally, BL produced on Day 8 in the absence (control) and presence of 0.1 mM LE were used for transferase-mediated dUTP nick end labeling and differential staining to evaluate, respectively the apoptotic rate and the allocation of cells into inner cell mass (ICM) and trophectoderm lineages (experiment 3). Despite similar blastocyst yields, supplementation of culture medium with 0.1 mM LE improved the cryotolerance of in vitro-produced (IVP) embryos compared to the control group, as indicated by higher (P < 0.05) hatching rates recorded after 48-hour post-warming culture (48.5%, 50.0%, and 63.8%, respectively with 0, 0.05, and 0.1 mM LE). Interestingly, when embryos were cultured in the presence of 0.1 mM LE, the percentage of BL with the most physiological ICM:total cells ratio (20%-40%) increased (85.1 vs. 66.0%, P < 0.05), confirming a beneficial effect on embryo quality. Furthermore, 0.1 mM LE decreased (P < 0.01) both the average number (4.3 ± 0.2 vs. 9.1 ± 0.3) and the proportion (3.6 ± 0.3 vs. 8.1 ± 0.5) of apoptotic cells in BL compared to the control. In conclusion, the enrichment of bovine culture medium with 0.1 mM LE improves embryo quality, as indicated by the improved cryotolerance, the lower apoptotic rate, and the higher percentage of BL with the most physiological ICM:total cells ratio.