A. Salzano

Effect of resveratrol supplementation during culture on the quality and cryotolerance of bovine in vitro produced embryos

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<0.01) and hatching rates (58.9% vs 30.9%, respectively; P<0.01) recorded after 48h post-warming culture. Blastocysts produced in the control and in 0.5μM resveratrol groups had similar numbers of ICM (34.1 and 36.4, respectively), TE (88.1 and 85.3, respectively) and total (122.2 and 121.7, respectively) cells. In conclusion, low levels of resveratrol during in vitro culture improve the quality of IVP bovine embryos, as indicated by their increased resistance to cryopreservation.

Nitrogen and phosphorus utilisation and excretion in dairy buffalo intensive breeding

The aim of this study was to quantify nitrogen (N) and phosphorus (P) utilisation and excretion in intensive dairy buffaloes. Italian Mediterranean buffaloes were divided into 4 groups (n=6/group) as follows: Groups M50, M125 and M225 at 50, 125 and 225 days of lactation, respectively, and Group NL which was non-lactating. Lactating buffaloes had greater (P<0.05) dry matter and organic matter intake than NL buffaloes. Buffaloes in Group M50 had a lower (P<0.05) crude protein (CP) apparent digestibility than buffaloes in Groups M125 and M225. Group NL had a higher (P<0.01) real CP digestibility than Group M50. Buffaloes in Groups M50 and NL had a negative N balance (-79 and -12 g, respectively) whilst buffaloes in Groups M125 and M225 had a positive N balance (26 and 16 g, respectively). Buffaloes in Group M50 had more (P<0.05) N in urine (204 g) than Group NL (87 g). Buffaloes in Group NL had less (P<0.01) P in both faeces (12.9 g) and urine (0.8 g) compared with the three groups of lactating buffaloes combined (25 g in faeces and 12 g in urine) and they also had greater (P<0.05) P digestibility (68%) than lactating buffaloes combined (43%). The present study has shown that buffaloes have a relatively high efficiency of N and P utilisation.

Genome-wide association studies to identify quantitative trait loci affecting milk production traits in water buffalo

Water buffalo is the second largest resource of milk supply around the world, and it is well known for its distinctive milk quality in terms of fat, protein, lactose, vitamin, and mineral contents. Understanding the genetic architecture of milk production traits is important for future improvement by the buffalo breeding industry. The advance of genome-wide association studies (GWAS) provides an opportunity to identify potential genetic variants affecting important economical traits. In the present study, GWAS was performed for 489 buffaloes with 1,424 lactation records using the 90K Affymetrix Buffalo SNP Array (Affymetrix/Thermo Fisher Scientific, Santa Clara, CA). Collectively, 4 candidate single nucleotide polymorphisms (SNP) in 2 genomic regions were found to associate with buffalo milk production traits. One region affecting milk fat and protein percentage was located on the equivalent of Bos taurus autosome (BTA)3, spanning 43.3 to 43.8 Mb, which harbored the most likely candidate genes MFSD14A, SLC35A3, and PALMD. The other region on the equivalent of BTA14 at 66.5 to 67.0 Mb contained candidate genes RGS22 and VPS13B and influenced buffalo total milk yield, fat yield, and protein yield. Interestingly, both of the regions were reported to have quantitative trait loci affecting milk performance in dairy cattle. Furthermore, we suggest that buffaloes with the C allele at AX-85148558 and AX-85073877 loci and the G allele at AX-85106096 locus can be selected to improve milk fat yield in this buffalo-breeding program. Meanwhile, the G allele at AX-85063131 locus can be used as the favorable allele for improving milk protein percentage. Genomic prediction showed that the reliability of genomic estimated breeding values (GEBV) of 6 milk production traits ranged from 0.06 to 0.22, and the correlation between estimated breeding values and GEBV ranged from 0.23 to 0.35. These findings provide useful information to understand the genetic basis of buffalo milk properties and may play a role in accelerating buffalo breeding programs using genomic approaches.

Evaluation of factors involved in the failure of ovum capture in superovulated buffaloes

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.

Integrating RNA-seq and GWAS reveals novel genetic mutations for buffalo reproductive traits

Genome-wide association study (GWAS) has been applied in buffalo breeding programs and been used to identify a number of candidate genes associated with buffalo reproductive traits. The genetic code of specific genes underlying buffalo reproductive traits remains unclear. Association study that measures both genetic and transcriptional variation has been applied for the investigation of complex traits. To investigate genes involved in buffalo reproductive traits, integrated RNA-seq results were investigated of buffalo granulosa cells and candidate genes which were reported to be associated with buffalo reproductive traits in a previous GWAS. A large number of variants were detected by RNA-seq, and 214 variants were located within the buffalo reproductive candidate genes identified by GWAS. A further association study in 462 Italian Mediterranean buffalo indicated that 25 SNPs distributed in 13 genes were associated with reproductive traits. Of the 13 genes, 11 were expressed in granulosa cells of all antral follicle development stages, and significant difference was found in the expression of NDUFS2 between follicles of diameter <8 mm and > 8 mm. These findings extend the results of GWAS by expanding the knowledge about new and potentially functional single-nucleotide polymorphisms and provide useful information about regulatory genes affecting buffalo reproductive traits.

164 CARNITINE IMPROVES POST-THAWING SPERM MOTILITY BY INCREASING ADENOSINE TRIPHOSPHATE CONTENT IN BUFFALO (Bubalus bubalis)

Semen cryopreservation plays a critical role for a wide application of both AI and in vitro embryo production in buffalo. In this species, spermatozoa are more susceptible to hazards during freezing and thawing than cattle spermatozoa, thus resulting in lower fertilizing potential (Andrabi et al. 2008 Anim. Reprod. Sci. 104, 427-433). Carnitine is a quaternary ammonium compound with antioxidant capacities, able to reduce the availability of lipids for peroxidation by transporting fatty acids into the mitochondria for β-oxidation to generate adenosine triphosphate (ATP) energy (Tanphaichitr and Leelahagul 1993 Nutrition 9, 246-54). It is known that cryopreservation processes decreases the intracellular concentration of carnitine in spermatozoa (Reyes-Moreno et al. 2000 J. Androl. 21, 876-86). In cattle, supplementation of semen extender with carnitine improves sperm motility and DNA integrity (Bucak et al. 2010 Cryobiology 61, 248-53). The aim of this study was to evaluate whether supplementation of semen extender with carnitine would increase ATP content in buffalo sperm and affect post-thawing motility. Eight ejaculates from 4 bulls were used for the trial. Each ejaculate was split into 3 equal aliquots and diluted at 37°C with BioXcell extender containing 0 (control), 2.5, and 7.5mM carnitine to a final concentration of 30×106 spermatozoa/mL. After 4h at 4°C, the straws were frozen in an automated system. At thawing, sperm motility was evaluated by phase contrast microscopy at 40× magnification (Gillan et al. 2008 Anim. Reprod. Sci. 103, 201-204). Adenosine triphosphate content was measured using a Colourimetric ATP Assay Kit (Biovision, Milpitas, CA, USA). Briefly, Percoll-separated spermatozoa were homogenised and then deproteinized using 10-kDa spin columns. Samples were incubated at RT for 30min and absorbance was measured at 570 nM in a microplate reader. Differences in sperm motility and ATP content among groups were analysed by ANOVA. Both concentrations of carnitine increased post-thawing sperm motility compared with the control (44.4±3.5, 53.1±3.9, and 52.5±3.6, respectively, with 0, 2.5, and 7.5mM carnitine; P<0.05). Interestingly, carnitine increased ATP content of buffalo frozen-thawed sperm in a dose-dependent manner (4.1±0.1, 5.3±0.1, and 8.2±0.4 nM×108 sperm, respectively, with 0, 2.5, and 7.5mM carnitine; P<0.01). In conclusion, the enrichment of semen extender with carnitine improved post-thawing motility of buffalo sperm by boosting mitochondrial ATP production, hence providing energy for use by spermatozoa.

51 CASPASE-3 INHIBITOR Z-VAD-FMK ENHANCES CRYOTOLERANCE OF IN VITRO-PRODUCED BOVINE PRE-IMPLANTATION EMBRYOS

In vitro-produced (IVP) bovine embryos are still less viable and resistant to cryopreservation than their in vivo counterparts. Cryopreservation induces cell degeneration through the apoptotic pathway in bovine oocytes and embryos (Men et al. 2003 Cryobiology 47, 73-81). Apoptosis can be prevented by inhibition of caspase activity, leading to improved cryosurvival in mammalian cells (Stroh et al. 2002 FASEB J. 16, 1651-3). Interestingly, cryotolerance of porcine embryos was improved by inhibiting apoptosis using a caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) during vitrification and subsequent culture (Men et al. 2006 Theriogenology 66, 2008-16). Aim of this work was to evaluate whether cryotolerance of bovine IVP embryos may be improved by using Z-VAD-FMK during cryopreservation and post-warming in vitro culture. Abattoir-derived bovine oocytes (n=753, over 4 replicates) were in vitro matured and fertilized according to standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347-55). Twenty hours after IVF, presumptive zygotes were cultured in SOF medium at 39°C with 5% CO2, 7% O2, and 88% N2. On Day 7, embryo yields were assessed and blastocysts (except the hatched blastocysts) were randomly divided in 2 groups: vitrification and post-warming culture in presence (n=60) or absence (n=54) of 20µM Z-VAD-FMK. Vitrification was carried out by Cryotop in 16.5% ethylene glycol, 16.5% DMSO, and 0.5M sucrose (Rubessa et al. 2011 Theriogenology 76, 1347-55). Blastocysts were warmed in decreasing sucrose solutions (0.25M for 1min and 0.15M for 5min) and cultured for 2 days. Resistance to cryopreservation was evaluated by assessing the survival rate, based on morphological criteria and hatching rate after 48h culture. Furthermore, TUNEL staining was used to evaluate the total cell (TC) number and the apoptotic rate of vitrified blastocysts after 48-h post-warming culture. Differences between groups in survival and hatching rates after 48-h post-warming culture were analysed by Chi-squared test, whereas differences in TC number and in number and percentage of apoptotic cells were analysed by Students t-test. Inhibition of caspase activity induced by Z-VAD-FMK increased embryo cryotolerance, as indicated by higher survival (92.6v. 55.0%; P<0.01) and hatching rates (40.7v. 23.3%; P<0.05) after 48h of post-warming culture. Furthermore, Z-VAD-FMK decreased both the average number (7.1±0.6v. 4.2±0.3; P<0.01) and the percentage (6.3±0.6v. 3.0±0.2; P<0.01) of apoptotic cells in blastocysts. No differences were recorded in TC number between groups (on average, 128.90±1.6). These results suggest that addition of 20µM Z-VAD-FMK during vitrification/warming and post-warming culture significantly inhibits apoptosis (DNA fragmentation) and improves the cryotolerance of IVP bovine embryos.

An association analysis between PRL genotype and milk production traits in Italian Mediterranean river buffalo

This Research Communication describes the association between genetic variation within the prolactin (PRL) gene and the milk production traits of Italian Mediterranean river buffalo (Bufala mediterranea Italiana). High resolution melting (HRM) techniques were developed for genotyping 465 buffaloes. The association of genetic polymorphism with milk production traits was performed and subsequently the effects of parity and calving season were evaluated. Single nucleotide polymorphisms (SNPs) were identified at exons 2 and 5 and at introns 1 and 2. All the SNPs were in Hardy-Weinberg equilibrium, and statistical analysis showed that the polymorphism of intron1 was significantly (P < 0·05) associated with milk yield, milk protein content and peak milk yield. The average contribution of the intron1 genotype (r 2 intron1) to total phenotypic variance in milk production traits was 0·09, and the TT genotype showed lower values than CC and CT genotypes. A nonsynonymous SNP was identified in exon 2, which resulted in an amino acid change from arginine to cysteine. Moreover, the polymorphism of exon 2 was associated significantly with milk fat content (P < 0·05), and the buffaloes with TT genotype showed higher total fat content than the buffaloes with CT genotype. These findings provide evidence that polymorphisms of the buffalo PRL gene are associated with milk production traits and PRL can be used as a candidate gene for marker-assisted selection in Italian Mediterranean river buffalo breeding.

Influences of different space allowance on reproductive performances in buffalo

The aim of this study was to evaluate the effect of two different conditions of space allowance on reproductive performance and oxidative parameters, biochemical and hormonal profiles in buffalo. The trial was carried out on one hundred pluriparous buffaloes divided into two different groups. Buffaloes in group HDG (high density group - n = 50) were maintained in open yards that allowed 10 m2 /head while those in group LDG (low density group - n = 50) were maintained in 22 m2 /head. After 60 days, 45 buffaloes in each group underwent synchronization of ovulation by Ovsynch and were artificially inseminated to assess the reproductive efficiency. On the day of AI blood samples were collected to evaluate oxidative stress, hormonal and metabolic profile. Furthermore, on the same day, blood, saliva and hair samples were collected to assess cortisol levels. Simultaneously, five buffaloes/group, were synchronized but not inseminated and on the day of the hypothetical timed artificial insemination (TAI), follicular fluid was recovered by OPU and blood samples were collected from each animal to evaluate the redox status on both plasma and follicular fluid. Conception rate on day 70 post-AI was similar between the two groups (57.5 vs. 62.5%, in LDG and HDG, respectively). No significant differences were found on redox status, metabolic and hormonal profile and cortisol levels between the groups. In conclusion, on the conditions of this experiment it was observed that the space allowance of 10 m2 /head did not affect reproductive efficiency in buffalo cows.

88 ENRICHMENT OF CULTURE MEDIUM WITH CROCETIN IMPROVES IN VITRO EMBRYO DEVELOPMENT IN CATTLE

The high incidence of developmental failure of bovine in vitro-produced embryos is due to suboptimal culture conditions that induce oxidative stress. Indeed, increased oxidative stress is one of the main factors affecting in vitro mammalian embryo development, decreasing the viability of IVP embryos. It is known that saffron has a powerful antioxidant capacity, mainly due to its active components crocin and crocetin. The aim of this study was to evaluate whether enriching the in vitro culture medium with crocetin improves in vitro embryo production efficiency in cattle. The range of concentrations of crocetin was chosen after a preliminary dose response trial (322 total presumptive zygotes were cultured with 0, 1, 10, and 50 μM, over 2 replicates) that showed beneficial and deleterious effects, respectively, with the lowest and highest concentration compared with the control (36.6 ± 5.6, 57.4 ± 4.5, 46.4 ± 4.4, and 6.8 ± 3.7% blastocyst rates, respectively, with 0, 1, 10, and 50 μM; P < 0.01). Therefore, the range of concentrations to test was reduced. Abattoir-derived bovine oocytes (n = 832, over 4 replicates) were in vitro matured and fertilized according to standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347–1355). Twenty hours after IVF, presumptive zygotes were cultured in SOF medium with 0 (control; n = 208), 1 μM (n = 208), 2.5 μM (n = 208), and 5 μM (n = 208), at 39°C under humidified air with 5% CO2, 7% O2, and 88% N2. The embryos obtained by the end of culture (i.e. on Day 7 post-IVF) were scored for quality, based on morphological criteria, and for developmental stage, as previously described (Robertson and Nelson 2010, Manual of the IETS, 86–105). The percentages of total transferable embryos and grade 1 and 2 blastocysts were recorded. As the chronology of development is a reliable parameter to assess quality, the percentage of fast-developing embryos (i.e. hatched and expanded blastocysts) was also compared among groups. Differences among groups were analysed by ANOVA, and Tukey method was used as a post-hoc test. Data are presented as means ± s.d. The supplementation of crocetin during culture did not affect cleavage rate (74.9 ± 6.3, 76.4 ± 8.4, 81.4 ± 4.3, and 76.4 ± 8.4%, respectively, with 0, 1, 2.5, and 5 μM). However, post-fertilization embryo development improved with 1 µM crocetin compared with the control, both in terms of total embryo output (43.8 ± 4.4, 61.1 ± 5.2, 50.4 ± 6.7, and 53.3 ± 7.3%, respectively, with 0, 1, 2.5, and 5 μM; P < 0.01) and grade 1 and 2 blastocysts (41.0 ± 3.6, 54.3 ± 5.4, 46.2 ± 6.7, and 49.4 ± 6.5%, respectively, with 0, 1, 2.5, and 5 μM; P < 0.05), whereas no differences were observed among the other groups. Moreover, the percentage of fast developing embryos increased with 1 µM (P < 0.05) crocetin compared with the control, with no other differences recorded among groups (17.7 ± 5.8, 34.7 ± 5.7, 24.9 ± 5.1, and 28.7 ± 7.8%, respectively, with 0, 1, 2.5, and 5 μM). In conclusion, these results demonstrated a beneficial effect of low concentrations of crocetin (1 μM) during culture both on blastocyst yield and quality, as indicated by the improved chronology of embryo development.