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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. [ABSTRACT FROM AUTHOR]

Simple Summary: In goat breeding, artificial insemination has a strong economic impact: It provides genetic improvement in intensive production systems and guarantees breed preservation in extensive systems. Sperm freezability is affected by several factors, such as breed species and freezing procedures. In addition, in goats, the seminal plasma enzyme phospholipase A reacts with egg yolk (component of semen extender), compromising sperm viability. Thus, seminal plasma is removed before freezing. This removal causes a decreasing level of antioxidants that prevent formation of toxic lipid peroxides with deleterious effects on fertility. Crocin is a water-soluble carotenoid that acts as an antioxidant, protecting cells and tissues against oxidation. Therefore, the aim of the present study was to assess the effect of crocin in the semen extender before cryopreservation. Five different goat breeds (Garganica, Jonica, Maltese Mediterranean Red and Saanen) were chosen to evaluate sperm qualitative characteristics, such as post-thawing sperm motility, viability, morphology and membrane integrity, as well as DNA fragmentation and intracellular ROS levels. The results highlighted that crocin supplementation in the extender decreased oxidative stress and improved sperm motility and the DNA integrity of frozen-thawed sperm 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract 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 E 2 and P 4 concentrations decreased in superovulated group (63.2 ± 10.6 vs 30.3 ± 5.9 ng/mL of E 2 and 130.1 ± 19.8 vs 71.6 ± 8.5 ng/mL of P 4 , 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. Highlights • Poor embryo recovery in superovulated buffalo is due to failure in ovum capture. • MOET impairs stereidogenesis and in turn ovarian and oviduct function in buffalo. • Exogenous FSH treatment affects gene expression in granulosa cells and oviduct. [ABSTRACT FROM AUTHOR]

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 × 10 8 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 × 10 6 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. [ABSTRACT FROM AUTHOR]

The influence of protein polymorphism on curd yield from the milk of 16 Italian Brown (IB) and 18 French Holstein (FH) primiparous cows was evaluated. The cows were bred on the same farm and fed with the same ration ad libitum. The chemical composition and protein polymorphism were determined in individual milk samples. The amount of real curd yield was monitored at 72 h and evaluated by micro cheese-making. For the same κ-CN genotype, FH primiparous cow produced more milk (+36.13%) with lower protein content than IB primiparous cow, resulting in a lower curd yield. IB primiparous cows presented a higher frequency of κ-CN BB genotype and preserved a better body condition score (BCS) (+0.57 BCS points P < 0.0008) at the end of lactation. [ABSTRACT FROM AUTHOR]

Simple Summary: The high embryo production cost in buffalo, compared to cattle, is currently the major factor limiting the commercial application of ovum pick-up and in vitro embryo production technologies. This is mainly due to the lower number of follicles that develop during each estrous cycle and consequently oocytes recovered per session. In this work, we tested two different hormonal treatments based on commercial follicle-stimulating hormone (FSH) and progesterone (P4) to enhance the number of follicles and competent oocytes. Furthermore, in the second experiment, we tested three different coasting times, i.e., periods between the last FSH administration and ovarian aspiration, which is known be the key period for the final oocyte maturation and competence acquisition. The results, in terms of number of aspirated follicles, oocyte quality, blastocyst yield, and superior quality blastocyst yield, allow us to suggest the use of high doses of FSH (40 mg given six times every 12 h) combined with the shorter coasting time (28–32 h) as the ideal protocol for ovarian superstimulation in buffalo. 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. [ABSTRACT FROM AUTHOR]