Establishment of effective and safe recipient preparation for germ-cell transplantation with intra-testicular busulfan treatment in pre-pubertal Barbari goats.

The busulfan, an alkylating agent, suppresses endogenous spermatogenesis in recipient testes. However, considering a wide variation in the effects of busulfan among animal species, its dosage and route of infusion need optimization to prepare effective and safe recipients. Thus, the current study aimed to create a suitable recipient goat model for germ cell (Gc) transplantation through a single intra-testicular (i.t.) busulfan infusion under ultrasonographic (USG) guidance. As observed through the infusion of trypan blue under USG guidance into mediastinum testis (MT) of pre-pubertal Barbari bucks, 3-5 mL of trypan blue solution could fill almost 80% of seminiferous tubules. Thereafter, in Experiment-1, the effect of different busulfan doses (mg/kg) i.e. 0 [negative control, Group (Gr) 1; 0 mg/kg-MT], 1 (Gr 2; 1 mg/kg-MT), 2 (Gr 3; 2 mg/kg-MT), and 3 (Gr 4; 3 mg/kg-MT) were studied. Further, in Experiment-2, sterilizing effects of busulfan infusion through two different routes [MT or cavum vaginale (CV)] were compared. Following i.t. busulfan treatment, no adverse physiological effects or body weight loss were detected. The histological analyses demonstrate a dose-dependent depletion of Gc with almost complete loss of Gc and spermatogenic activities in Gr 3 and 4, and extensive fibrosis in Gr 4. A considerable suppression of spermatogenesis marked with devoid of endogenous spermatogonial population and absence of significant (P > 0.05) effect on key hematological variables were observed in 2 mg/kg-MT Gr. These findings coupled with the results of significant (P < 0.05) down-regulation of marker genes of undifferentiated spermatogonia (THY-1 and PLZF), Gc pluripotency (UCHL-1, OCT-4, and DDX-4), and adhesion (E-cadherin and ß-integrin); up-regulation of apoptotic genes (ID - 4 and BCL-6), and unchanged expression of Sertoli cell marker (vimentin), confirmed the safe and efficient depletion of endogenous Gc in 2 mg/kg-MT Gr. Furthermore, the effect of busulfan infusion on scrotal-testicular biometry, endocrine variables (plasma cortisol and testosterone), and Gc removal was more evident when busulfan was infused into MT than into CV. Overall, the results demonstrated that 2.0 mg/kg is an optimal single dose of busulfan when infused into the MT under USG guidance for the preparation of pre-pubertal recipient bucks. Overall, this study provides a basis to prepare suitable recipients through providing an available niche for efficient Gc transplantation in goats.

Effects of unconjugated gold, silver and titanium dioxide nanoparticles on bovine spermatozoa at various stages of cryopreservation.

BACKGROUND: The increasing use of nanoparticles (NP) for gender-selected spermatozoa, sperm-enriched semen and novel extenders raises the concern of undesirable effects on fertility and sperm function. OBJECTIVE: To investigate the effects of gold (Au-), silver (Ag-), and titanium dioxide (TiO2-) NPs on the motility and sperm functions in bovine spermatozoa at various stages of cryopreservation. MATERIALS AND METHODS: Frieswal (Sahiwal × Holstein Friesian) bull semen ejaculates (N = 24) were challenged with unconjugated and ligand-free Au-, Ag-, and TiO2-NPs. RESULTS: At post-dilution (fresh) stage, there was no significant difference observed in progressive motility and viability amongst the control and any nanoparticle-treated groups, though plasma membrane integrity was significantly reduced in nanoparticle-treated groups (p < 0.05). The acrosome intactness was also significantly reduced in the groups of Ag-NP and TiO2 -NP (p < 0.05), while there was no effect observed in the Au-NP group. At post-equilibration stage, a significant reduction in motility, viability, and plasma membrane integrity was observed in all three nanoparticle-treated groups (p < 0.05). There was no difference in intact acrosome between the control and Au-NPs groups; which was significantly higher than the Ag-NP and TiO2 -NP groups (p < 0.05). At post-thaw stage, all NP groups resulted in a significant reduction of motility, viability, acrosome intactness and plasma membrane integrity (p < 0.05). Besides, TiO2-NPs appear to be significant more toxic (p < 0.05) among three NP groups, and Au-NPs appear to be lesser toxic. CONCLUSION: Bovine spermatozoa are adversely affected by Au-, Ag- and TiO2 -NPs that may impair sperm motility and other functions. doi.org/10.54680/fr22310110512.

Supplementation of Slow-Release Melatonin Improves Recovery of Ovarian Cyclicity and Conception in Summer Anoestrous Buffaloes (Bubalus bubalis).

The role of melatonin as a protective neurohormone against restoring cyclicity in summer anoestrous animals in photoperiod species has gained wider acceptance. This study was designed to uncover the evidence the slow-release melatonin (MLT) has on initiation of ovarian cyclicity and conception rate (CR) in summer anoestrous buffaloes. Thus, buffaloes diagnosed as summer anoestrous (absence of overt signs of oestrus, concurrent rectal examination and radioimmunoassay for serum progesterone at 10 days interval) were grouped as untreated (Group I, sterilized corn oil, n = 8) and treated (Group II, single subcutaneous injection of MLT @18 mg/50 kg bwt in sterilized corn oil, n = 20). Animals treated and detected in oestrus were artificially inseminated (AI) followed by division into Group III (second dose of MLT on 5th day post-AI, n = 8) and Group IV (no melatonin administration, n = 10). Blood samples were collected at 4 days interval for estimation of serum MLT, progesterone and oestrogen using radioimmunoassay kit. Mean oestrous induction rate (OIR), oestrous induction interval (OII), interoestrous interval (IOI) and CR were estimated. Compared to control, concentration of melatonin was significantly (p < 0.05) higher in treated group ranging from 14.34 ± 1.72 to 412.31 ± 14.47 pg/ml whereas other two hormones did not show any concentration difference. Melatonin-administered buffaloes showed significantly (p < 0.05) higher (90%) OIR with OII of 18.06 ± 1.57 days. Results showed improvement in conception rate in buffaloes administered with post-insemination melatonin. It can be concluded from the study that slow-release melatonin supplementation restored cyclicity in summer anoestrous animals resulting in improvement in conception rate in buffaloes.

Sustained delivery of exogenous melatonin influences biomarkers of oxidative stress and total antioxidant capacity in summer-stressed anestrous water buffalo (Bubalus bubalis).

High ambient temperature during summer in tropical and subtropical countries predisposes water buffaloes (Bubalus bubalis) to develop oxidative stress having antigonadotropic and antisteroidogenic actions. Melatonin is a regulator of seasonal reproduction in photoperiodic species and highly effective antioxidant and free radical scavenger. Therefore, a study was designed to evaluate the effect of sustained-release melatonin on biomarkers of oxidative stress i.e., the serum malondialdehyde (MDA) and nitric oxide (NO), and the total antioxidant capacity (TAC). For the study, postpartum buffaloes diagnosed as summer anestrus (absence of overt signs of estrus, concurrent rectal examination, and RIA for serum progesterone) were grouped as treated (single subcutaneous injection of melatonin at 18 mg/50 kg body weight dissolved in sterilized corn oil as vehicle, n = 20) and untreated (subcutaneous sterilized corn oil, n = 8). Blood sampling for estimation of serum TAC and MDA (mmol/L) and NO (µmol/L) was carried out at 4 days of interval from 8 days before treatment till 28 days after treatment or for the ensuing entire cycle length. Results showed serum TAC concentration was higher in the treatment group with a significant (P < 0.05) increasing trend, whereas MDA and NO revealed a significant (P < 0.05) decline. Serum MDA and NO were higher in control compared with those of treatment group. Moreover, buffaloes in the treatment group showed 90% estrus induction with 18.06 ± 1.57 days mean interval from treatment to the onset of estrus. These results report that melatonin has a protective effect by elevating antioxidant status and reducing oxidative stress resulting in the induction of cyclicity in summer-stressed anestrous buffaloes.