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.

Evaluation of bovine sperm telomere length and association with semen quality.

The study aimed to evaluate if the sperm telomere length can be considered as a new biomarker for sperm quality in bulls. Sperm Telomere Length was evaluated by Monochrome Multiplex Quantitative PCR in group A (n = 8) and group B (n = 8) bulls, classified according to standard semen analysis. Also, this parameter was measured before and after Percoll gradient separation within bulls that produced semen of satisfactory quality. Sperm telomere length, measured as T/S ratio (average ratio of telomere repeats copy number to a single copy gene), was higher in group A than in group B bulls (0.77 ± 0.03 vs 0.43 ± 0.06; P < 0.01). Sperm telomere length was positively correlated with motility, viability and membrane integrity, and it was negatively correlated with sperm anomalies. Furthermore, Percoll gradient selected sperms with higher T/S ratio than unselected sperms (1.19 ± 0.02 vs 0.67 ± 0.03). These results suggest that sperm telomere length can be used as a new marker of bovine semen quality.

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.

Cholesterol-loaded cyclodextrins prevent cryocapacitation damages in buffalo (Bubalus bubalis) cryopreserved sperm.

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.