Accounting for Genetic Differences Among Unknown Parents in Bubalus bubalis: A Case Study From the Italian Mediterranean Buffalo.

The use of genetic evaluations in the Water Buffalo by means of a Best Linear Unbiased Prediction (BLUP) animal model has been increased over the last two-decades across several countries. However, natural mating is still a common reproductive strategy that can increase the proportion of missing pedigree information. The inclusion of genetic groups in variance component (VC) and breeding value (EBV) estimation is a possible solution. The aim of this study was to evaluate two different genetic grouping strategies and their effects on VC and EBV for composite (n = 5) and linear (n = 10) type traits in the Italian Mediterranean Buffalo (IMB) population. Type traits data from 7,714 buffalo cows plus a pedigree file including 18,831 individuals were provided by the Italian National Association of Buffalo Breeders. VCs and EBVs were estimated for each trait fitting a single-trait animal model and using the official DNA-verified pedigree. Successively, EBVs were re-estimated using modified pedigrees with two different proportion of missing genealogies (30 or 60% of buffalo with records), and two different grouping strategies, year of birth (Y30/Y60) or genetic clustering (GC30, GC60). The different set of VCs, estimated EBVs and their standard errors were compared with the results obtained using the original pedigree. Results were also compared in terms of efficiency of selection. Differences among VCs varied according to the trait and the scenario considered. The largest effect was observed for two traits, udder teat and body depth in the GC60 genetic cluster, whose heritability decreased by -0.07 and increased by +0.04, respectively. Considering buffalo cows with record, the average correlation across traits between official EBVs and EBVs from different scenarios was 0.91, 0.88, 0.84, and 0.79 for Y30, CG30, Y60, and CG60, respectively. In bulls the correlations between EBVs ranged from 0.90 for fore udder attachment and udder depth to 0.96 for stature and body length in the GC30 scenario and from 0.75 for udder depth to 0.90 for stature in the GC60 scenario. When a variable proportion of missing pedigree is present using the appropriate strategy to define genetic groups and including them in VC and EBV is a worth-while and low-demanding solution.

Early Prediction of Corpus Luteum Functionality Using an Imaging Software.

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.

FSH Stimulation with Short Withdrawal Improves Oocyte Competence in Italian Mediterranean Buffalo (Bubalus bubalis).

The aim of this work was to evaluate the efficiency of different FSH doses and FSH coasting times before ovum pick-up (OPU) on follicular growth and oocyte competence in buffalo. Experiment 1 involved two different FSH treatments: 40 mg FSH given three (FSH3) or six (FSH6) times, 2 days after dominant follicle removal were tested, with OPU carried out after 40-44 h of coasting. In experiment 2, OPU was carried out after FSH6 protocol followed by 28-32 h (C1), 40-44 h (C2), or 64-68 h (C3) of coasting time. Cumulus oocyte complexes (COCs) were classified, in vitro matured, fertilized, and cultured. The results demonstrated that FSH6 increased the total number of follicles, the number and percentages of medium and large follicles, the number and the proportion of good quality oocytes, and the number of grade 1,2 and fast-developing blastocysts compared to the control. C3 decreased the percentage of good quality oocyte and blastocyst rates compared to C1 and C2. A higher percentage of fast blastocysts and average number of grade 1,2 blastocysts was observed in C1 compared to C3, with intermediate values found in C2. The improved efficiency in terms of blastocyst yields suggests the use of FSH6 + C1 protocol for ovarian superstimulation in buffalo.

Analysis of meiotic segregation by triple-color fish on both total and motile sperm fractions in a t(1p;18) river buffalo bull.

Chromosomal aberrations are relatively frequent pathologies in both humans and animals. Among them, translocations present a specific meiotic segregation pattern able to give a higher percentage of unbalanced gametes that can induce fertility problems. In this study, the meiotic segregation patterns of 1p, 1q and 18 Bubalus bubalis chromosomes were analyzed in both total sperm fraction and motile sperm fraction of a t(1p;18) carrier and a control bulls by triple-color FISH analysis with a pool of specific BAC probes. The frequencies of each total sperm fraction products in the carrier resulting from alternate, adjacent I, adjacent II and 3:1 segregation were 39%, 20%, 1% and 38%, respectively. On the other hand, the frequencies of each motile sperm fraction products in the carrier resulting from alternate, adjacent I, adjacent II and 3:1 segregation were 93%, 5%, 0% and 2%, respectively. The frequencies of normal sperms in the carrier were 27% and 69% in total sperm fraction and motile sperm fraction, respectively. The frequencies detected in motile sperm fraction were also validated by comparison with bull's progeny. To our knowledge, this is the first report on the meiotic segregation patterns in motile sperm fractions of B. bubalis bull carrying a chromosomal translocation. These data suggest that translocation has a very limited effect on aneuploidy in the gametes, and therefore, on the reproductive abilities of the bull.

Crocin Improves the Quality of Cryopreserved Goat Semen in Different Breeds.

The effect of crocin in the semen extender before cryopreservation was evaluated on sperm parameters of 20 bucks of five different breeds: Garganica (GA), Jonica (JO), Maltese (MA), Mediterranean Red (MR) and Saanen (SA). Semen samples were centrifuged, to remove seminal plasma, divided in two aliquots and diluted with Tris-egg-yolk-based extender, containing 0 (control group) and 1 mM crocin. Crocin concentration was established after a preliminary dose trial. On fresh and frozen-thawed sperm, motility, viability, morphology, membrane integrity, DNA fragmentation and ROS levels were evaluated. The freezing process led to a decrease (p < 0.05) in all the sperm parameters recorded, confirming the deleterious effect of cryopreservation on goat semen. The most interesting result regarding the inclusion of crocin in the extender before cryopreservation was as follows: Crocin significantly improved (p < 0.05) sperm motility in all breeds, except for Mediterranean Red, compared to the control group. Furthermore, 1 mM crocin reduced percentage of spermatozoa with DNA fragmentation with a marked decrement (p < 0.05) in Garganica and Saanen, as compared to the control group. Finally, intracellular ROS decreased (p < 0.01) in the crocin-treated sperm of all breeds, as compared to the control. In conclusion, supplementation of 1 mM crocin in the extender decreased oxidative stress, improving sperm motility and the DNA integrity of frozen-thawed sperm in different breeds.

Inhibition of apoptosis by caspase inhibitor Z-VAD-FMK improves cryotolerance of in vitro derived bovine embryos.

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 µM 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 ± 0.3 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 µM 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.

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 ±â€¯10.6 vs 30.3 ±â€¯5.9 ng/mL of E2 and 130.1 ±â€¯19.8 vs 71.6 ±â€¯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.

Carnitine supplementation decreases capacitation-like changes of frozen-thawed buffalo spermatozoa.

The aim of this study was to evaluate the effect of carnitine supplementation of semen extender on fertility parameters of frozen-thawed buffalo sperm. Buffalo semen was cryopreserved in BioXcell containing 0 (control group), 2.5 and 7.5-mM carnitine. After thawing, viability, motility, membrane integrity and capacitation status (assessed by localization of phosphotyrosine-containing proteins and chlortetracycline, chlortetracycline assay) were evaluated. Furthermore, total antioxidant capacity, reactive oxygen species (ROS) and lipid peroxidation levels, as well as adenosine triphosphate (ATP) content and phospholipids concentration were assessed. Finally, in vitro-fertilizing ability was evaluated after heterologous IVF. An increased post-thawing sperm motility and membrane integrity were recorded in both treated groups compared with the control (44.4 ± 3.5, 53.1 ± 3.9, and 52.5 ± 3.6%; P < 0.05 and 48.44 ± 0.69, 55.19 ± 0.54, 59.63 ± 0.30%; P < 0.01 with 0, 2.5-mM, and 7.5-mM carnitine, respectively). Supplementation of carnitine to the freezing extender decreased (P < 0.01) the percentage of sperm displaying fluorescence at both equatorial and anterior acrosomal regions (pattern EA), corresponding to high capacitation level, compared with the control (30.3 ± 3.8, 18.8 ± 2.8, and 7.2 ± 2.9%, respectively, with 0, 2.5-mM, and 7.5-mM carnitine). In agreement with this, carnitine also decreased (P < 0.01) the percentage of sperm displaying chlortetracycline pattern B (capacitated sperm) (63.8 ± 1.8, 46.8 ± 2.2, and 37.2 ± 1.8%, respectively with 0, 2.5-, and 7.5-mM carnitine). Interestingly, carnitine increased total antioxidant capacity and ATP content of buffalo frozen-thawed sperm (1.32 ± 0.02, 1.34 ± 0.01, 1.37 ± 0.01 mM/L and 4.1 ± 0.1, 5.3 ± 0.1 and 8.2 ± 0.4 nM × 108 sperm; P < 0.01, respectively, with 0, 2.5- and 7.5-mM carnitine). Intracellular ROS decreased in carnitine-treated sperm compared with the control, as indicated by dihydroethidium (DHE) values (0.22 ± 0.01, 0.18 ± 0.01, and 0.14 ± 0.0 µM/100 µL dihydroethidium, respectively, with 0, 2.5-, and 7.5-mM carnitine; P < 0.01), whereas lipid peroxidation levels (on average 30.5 ± 0.3 nmol/mL MDA) and phospholipids concentration (on average 0.14 ± 0.00 µg/120 × 106 sperm) were unaffected. Despite the improved sperm quality, the percentage of normospermic penetration after IVF was not influenced (on average 53.5 ± 1.8). In conclusion, enrichment of extender with carnitine improved buffalo sperm quality by increasing ATP generation and modulating ROS production, without affecting in vitro fertilizing ability.

Resveratrol prevents capacitation-like changes and improves in vitro fertilizing capability of buffalo frozen-thawed sperm.

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 < 0.01) the percentage of sperm displaying pattern A (low capacitation level), but treatment with 50-µM resveratrol also decreased (P < 0.01) the proportion of sperm exhibiting pattern EA (high-capacitation level) compared with the control. Interestingly, supplementation of semen extender with resveratrol increased membrane integrity, indicated by the higher percentage of hypo-osmotic swelling positive sperm (55.6 ± 0.6 vs. 48.4 ± 0.7; P < 0.01), and total antioxidant capacity (1.36 ± 0.01 vs. 1.32 ± 0.02 mM/L; P < 0.05) compared with the control. Intracellular reactive oxygen species decreased in resveratrol-treated sperm compared with the control, as indicated by dihydroethidium values (0.17 ± 0.01 and 0.22 ± 0.01 µM/µL dihydroethidium, respectively; P < 0.01). Moreover, when IVF was carried out by using semen treated with 50 µM resveratrol, the normal fertilization rate considerably improved (60.8%, P < 0.05) compared with the control (51.3%). However, no differences were recorded in pregnancy rates at 60 days post-AI with resveratrol-treated semen (50 µM) compared with the control (48.7 vs. 46.5%, respectively). In conclusion, the inclusion of 50-µM resveratrol in the extender decreases capacitation-like changes and oxidative stress, improving membrane stability and in vitro fertilizing ability of buffalo semen.