Molecular insights on skewing of sex ratio in rabbits (Oryctolagus cuniculus) supplemented with dietary calcium and magnesium.

The effect of dietary calcium (Ca) and magnesium (Mg) supplementation on serum biochemical parameters, steroid hormones, gene expression, and the sex ratio was investigated in female New Zealand white rabbits. A total of 25 rabbits were allocated into five treatment groups: The control group was fed with regular pellet feed, whereas, treatment groups were supplemented with Ca and Mg: T1 (0.40% and 0.01%), T2 (0.60% and 0.02%), T3 (0.80% and 0.03%) and T4 (1.00% and 0.04%), respectively. The rabbits were subjected to three breeding cycles. The T3 group skewed towards females (65.33%) from all three breeding. There was elevated Ca concentration in T3 (15.26 ± 0.77 mg dL-1) and T4 (15.61 ± 0.82 mg dL-1) groups compared to the control. The concentration of estradiol was significantly high in T3 and T4 groups at 0.5 days post-coitus (dpc) and T2, T3 and T4 groups at 21dpc. Testosterone was significantly high in T4 group at 0.50 dpc and T2 and T4 group at 21dpc. The expression of 13 genes was studied in the oviduct. Genes such as OVGP1, CCT4, ANXA2 and TLR4 were up-regulated and positively correlated with the female sex ratio. The molecular functions and pathways of up-regulated genes were suggestive of their role in fertilization such as sperm selection, sperm storage, immune regulation, implantation and early embryonic development. The variations in the serum electrolytes, steroid hormones and gene expression might have an impact on the skewing process.

In vitro production of desired sex ovine embryos modulating polarity of oocytes for sex-specific sperm binding during fertilization.

The present study aimed to modulate the oxidative status-mediated polarity of the oocytes for sex-specific sperm fertilization to generate desired sex embryos. In vitro embryos were produced at different oxidative status, varying O2 concentrations, and without/with L-carnitine in maturation and culture media. The majority of the embryos produced at high oxidative stress were males whereas; low oxidative status favoured female embryos production. Low O2 doubled the proportion of female embryos (10.59 vs 21.95%); however, L-carnitine supplementation in media increased approximately seven-folds of the female embryos (12.26 vs. 77.62%) production. Oocytes matured at high oxidative status were in the repolarized state favouring positively charged Y sperm fertilization to produce significantly more male embryos. Low oxidative status favoured negatively charged X sperm fertilization to the oocytes in the depolarized state to produce more female embryos. Intracellular ROS was significantly low in female embryos than in males; however, female embryos were more stressful than males. The study concluded that the oxidative status-mediated alteration in pH of the medium to modulate the intracellular positive ions is the main critical factor to influence the sex of embryos through sex-specific sperms fertilization to the oocytes as per their polarity.

Organic mineral supplementation on differential protein profile of Osmanabadi bucks (Capra hircus).

The present study aimed to determine the differential protein profile of seminal plasma proteins of bucks supplemented with trace minerals. Forty bucks of uniform size and body weight were assigned as ten groups (n = 4). The control group (T1) was fed with the control diet (concentration mixture and roughages) whereas the remaining groups were supplemented the control diet with Zn20 mg (T2), Zn40 mg (T3), Zn60 mg (T4), Cu12.5 mg (T5), Cu25 mg (T6), Cu37.5 mg (T7), Zn20 mg + Cu12.5 mg (T8), Zn40 mg + Cu25 mg (T9), and Zn60 mg + Cu37.5 mg (T10) for eight months. Seminal plasma proteins from each group were subjected to two-dimensional electrophoresis and fifteen differential proteins were selected based on differential expression, subjected to identification using Nano-LC-MS/MS (LTQ-Qrbitrap-MS). The identified proteins were Triacylglycerol lipase, EGF like repeats and discoidin domains 3, Lipocalin, Iodothyronine deiodinase, Transcription factor AP2-delta, 60S ribosomal protein L13, IST1 factor associated with ESCRT-III, Lysozyme, Uncharacterized protein (BRI3-binding protein), Uncharacterized protein, Histone deacetylase 11, General transcription factor IIF subunit 2, Nudix hydrolase 6, Protein kinase cAMP-activated catalytic subunit beta and Elongin C. The organic Cu supplemented group is the better than the organic Zn and organic Zn + Cu supplemented groups.

Interaction of apoptosis and pluripotency related transcripts for developmental potential of ovine embryos produced in vitro at different oxygen concentrations.

The present study in sheep model was to find out the interaction of apoptotic transcripts, that is, Bcl2, Bax, Casp3, PCNA and p53 and pluripotency related transcripts, that is, Sox2, Nanog and Oct4 in ovine embryos produced in vitro at different O2 concentrations (20% and 5% O2) to compare their developmental potential. Oxygen concentrations did not influence the maturation and cleavage rate but the percentage of morula and blastocysts was significantly more at 5% as compared to 20% O2. A significant upregulated expression of Bcl2 and PCNA genes and significantly downregulated expression of Casp3 and p53 were observed in the blastocysts at 5% than those at 20% O2. The expression of Bax was not influenced by the O2 concentration. Among the pluripotency related transcripts, the expression of Oct4 was significantly upregulated and the expression of Sox2 and Nanog was significantly downregulated in embryos at 5% than at 20% O2. The study concluded that the embryos produced in vitro at low O2 (5%) concentration regulate the expression of developmental genes related to apoptosis and pluripotency to improve the developmental potential of embryos as compared to high O2 (20%) concentration.

Cryoprotective role of organic Zn and Cu supplementation in goats (Capra hircus) diet.

The current study focused on cryopreservation and assessment of characters of post-thaw semen of indigenous Osmanabadi bucks maintained with standard diet, supplemented with different concentrations of organic zinc (Zn), copper (Cu) or in combination, for a period of 180 days. The different doses of organic Zn and Cu were fed per kg DM basis, Zn groups (low: Zn20, medium: Zn40 and high: Zn60), Cu groups: (low: Cu12.5, medium: Cu25 and high: Cu37.5) and combination of Zn + Cu groups (low: Zn20 + Cu12.5, medium: Zn40 + Cu25 and high: Zn60 + Cu37.5) respectively. The control group bucks were maintained mainly on the basal diet without any additional mineral supplementation. Two hundred and forty (240) semen samples were collected from 40 bucks aged 11 months, through electro ejaculator method, processed and analysed for sperm quality parameters both at pre freeze and post-thaw stage. The semen samples were diluted in Tris egg yolk extender, cooled and equilibrated for 4 h at 5 °C, cryopreserved using programmable freezer (PLANER Kryo 360-1.7) and stored at -196 °C. The organic trace minerals (Zn, Cu and Zn + Cu) protected the spermatozoa against the cryoinjury and maintained higher post-thaw semen parameters except in high Zn group. Additional feeding of organic Cu and Zn to bucks had a protective role and resulted in higher sperm liveability, plasma membrane and acrosome integrities, motility and velocity and reduced oxidative stress in supplemented goats (P < 0.05).

Expression of Apoptotic and Antioxidant Enzyme Genes in Sheep Oocytes and In Vitro Produced Embryos.

The present study was to find out the expression pattern and relative expression level of apoptotic (Bcl2, Bax, Casp3, and PCNA) and antioxidant enzyme [(GPx, Cu/Zn-SOD (SOD1) and Mn-SOD (SOD2)] genes in sheep oocytes and developing embryos produced in vitro by conventional RT-PCR and real time qPCR, respectively. Different developmental stages of embryos were produced in vitro from oocytes collected from local slaughter house ovaries. RT-PCR amplicons showed expression of Bcl2 and PCNA in all stages except at morula. In contrast Bax and Casp3 were expressed in all stages. GPx and SOD1 were expressed in all stages but SOD2 was not expressed in 8-16 cells, although expressed in the remaining stages. The qPCR analysis reflected that Bcl2 expression was significantly (P < 0.05) downregulated in morula and maximum upregulated expression was observed in in vitro matured oocytes. Higher upregulated expression (P < 0.05) of Bax was in morula and downregulated expression was at 2-4 cells. Casp3 was significantly upregulated at 8-16 cells and downregulated in in vitro matured oocyte. PCNA expression was highest at blastocyst and least expression was at morula. GPx was expressed significantly highest in matured oocytes and least expression was at zygote. SOD1 was expressed significantly highest at 8-16 cells and least expression was at zygote. Expression of SOD2 was least among all the antioxidant enzymes but significantly higher expression of SOD2 was in immature oocyte; however, least expression was at 8-16 cells. It can be concluded from the study that the sheep embryos produced in vitro are highly sensitive to culture condition, which alters the expression level of apoptotic and antioxidant enzyme genes.

GnRH-1 mRNA, LH surges, steroid hormones, egg production, and intersequence pause days alter in birds exposed to longer wavelength of light in the later stages of production in Gallus gallus domesticus.

The objective of this was to establish the effects of red spectrum of light (650 nm, treated n = 12) and normal spectrum of light (450 nm control = 12) on GnRH-I mRNA expression, amplitude and frequency of luteinizing hormone (LH), and egg production from 72-82 weeks of age in white leghorn hens. Birds exposed to red spectrum of wavelength significantly improved (P < 0.01) steroid hormone, and egg production improved over old laying 72 to 82 weeks. Weekly interval profiles followed the same pattern. At 77th weeks of age blood, samples from both the groups were collected at every 3 h for 36 h to study the pulsatile secretion of LH surges. Plasma LH concentration was higher (P < 0.01) in treated birds with more number of frequencies and amplitude LH surges in plasma of treated birds. LH frequencies were more pronounced and advanced during 36 h of sampling at 3 h interval in treated birds. Weekly interval of plasma LH, E2ß, and P(4) concentrations increased (P < 0.01) in treated birds from 72 to 82 weeks of age. GnRH-I mRNA concentration was significantly (P < 0.01) higher in birds exposed to red spectrum of light compared to controls. It is hypothesized that exposure of birds to red spectrum of light-enhanced (P < 0.01) GnRH-I mRNA with more number of yellow yolky follicles was found in birds exposed to red spectrum of light during 77 days (72-82 weeks of age) of experimental period. It is concluded that higher levels of GnRH-I mRNA, LH, E2ß, and P(4) concentration with lower incidence of pause days enabled the birds to lay more eggs even later in the productive period by modulating the wavelengths of light under normal husbandry conditions.