Induction,purification and partial characterization of vitellogenin in an Indian major carp Catla catla (Ham.)

Vitellogenin was purified from the serum of 17‐β estradiol (E₂)‐induced juvenile Catla catla using a simple two‐step purification procedure i.e. selective chemical precipitation followed by gel filtration chromatography. Purified protein migrated as single band in a native gradient PAGE which indicated the purity of the sample. The molecular weight of the native catla vitellogenin (~440 kDa) was determined using gel filtration chromatography. In SDS‐PAGE under reducing conditions catla vitellogenin dissociated into three major sub units at 115 kDa, 102 kDa and 73 kDa along with a few faint bands. Confirmation of purified protein as catla vitellogenin was supported by multiple physiological evidences, e.g. absence in male as well as juvenile sera and presence in matured female fish, ability to be synthesized upon estradiol injection in immature fish and certain unique biochemical properties like high molecular weight, phospholipoglycoprotein nature of the molecule. Western blot analysis showed that polyclonal antibody raised against purified protein detected vitellogenin in the sera of catla and in a few species selected from Cyprinidae family. These antisera were used to detect vitellogenin in liver tissue of hormone‐induced catla using immunohistochemistry and its applicability in other immunoassays is discussed.     

Viability and Growth of Buffalo Preantral Follicles and their Corresponding Oocytes In Vitro: Effect of Growth Factors and β Mercaptoethanol

The present study was undertaken to isolate buffalo preantral follicles (PFs), to test the viability and sizes of buffalo PFs and to examine the effect of various growth factors (insulin-like growth factor, fibroblast growth factor) and an antioxidant (β mercaptoethanol) on the in vitro growth, survival and antrum formation rates of buffalo PFs and growth rates of oocytes in cultured PFs. Preantral follicles from slaughtered buffalo ovaries were recovered by a combined mechanical and enzymatic method. The recovery rates of >40–100, 101–200, 201–300, 301–400 and 401–500 μm PFs were 5.1, 3.2, 3.1, 6.3 and 5.1 per ovary, respectively. The corresponding viability rates were 76.1%, 78.1%, 85.2%, 92.5% and 92.6%, respectively. There was a positive correlation ( r  = 0.73) between oocyte size and the follicular size. However, there was no significant correlation between the size of oocyte and its viability at the time of its retrieval from ovary. Insulin-like growth factor and fibroblast growth factor improved the survival of buffalo PFs and regulated their growth in culture. The growth factors and β mercaptoethanol in association synergically improved the growth and survival of buffalo PFs.     

Effect of supplementation of blue green alga (Spirulina) on outcome of pregnancy in rats

To study the supplementary effect of Spirulina, pregnant rats were fed 5 different kinds of diets (casein, Spirulina, wheat gluten, Spirulina + wheat gluten, Spirulina-without additional vitamins and minerals), each providing 22% protein during the period of pregnancy. The outcome of pregnancy was assessed from litter and dams' weight and litter size. Maternal weight gain was found to be maximum with Spirulina + wheat gluten and least with the wheat gluten diet. Rats receiving Spirulina containing diets produced significantly (p<0.05) higher litter size than those receiving casein and wheat gluten. In spite of having higher litter size, Spirulina containing diet groups produced pups with birth weights comparable to those of casein. Spirulina appears to be a good dietary supplement during pregnancy.     

Amelioration of oxidative stress using N‐acetylcysteine in canine parvoviral enteritis

Previously, antioxidants have not been evaluated for treatment of parvoviral diarrhea in dogs. In this study, antioxidant potential of N‐acetylcysteine (NAC) in dogs infected with canine parvovirus with a nonblinded randomized clinical trial has been carried out. A total 18 parvo‐infected dogs were randomly divided into two groups: nine parvo‐infected dogs were treated with supportive treatment and nine parvo‐infected dogs were treated with NAC along with supportive treatment. Simultaneously, nine healthy dogs were kept as healthy control. In parvo‐infected dogs, marked hemoconcentration, leucopenia, neutropenia and oxidative stress were noticed compared to healthy dogs. The NAC treatment progressively improved the leukocyte, neutrophil, monocyte, and eosinophil counts over the time in parvovirus‐infected dogs compared to dogs that received only supportive treatment. In addition, NAC treatment significantly improved glutathione S‐transferase (GST) activity and decreased nitrite plus nitrate (NOx) and malondialdehyde (MDA) concentrations on day 3 and 5 compared to supportive treatment in parvo‐infected dogs. However, supportive treatment alone failed to ameliorate oxidative stress in the infected dogs till day 5. The results of this study suggest that NAC represents a potential additional treatment option that could be considered to improve the health condition and minimize the duration of hospitalization in case of canine parvoviral diarrhea.     

Molecular cloning and expression of FGF2 gene in pre‐implantation developmental stages of in vitro‐produced sheep embryos

Early embryonic mortality is one of the main sources of reproductive loss in domestic ruminants including sheep. Fibroblast growth factor‐2 (FGF‐2) is a member of FGFs family that mediates trophoblast activities and regulates embryonic development in various species. In this study, we have cloned, characterized sheep FGF2 cDNA (KU316368) and studied the expression in sheep embryos. Ovaries of non‐pregnant sheep were collected from local abattoir and matured in culture medium at 38.5ºC, 5% CO₂, 95% humidity for 22–24 hr. The matured oocytes were inseminated with capacitated spermatozoa in Brackett and Oliphant medium and resulted embryos were cultured in CO₂ incubator for 6–7 days to complete the developmental stages from two cells to blastocyst stage. Total RNA was extracted from immature oocytes (n = 100), mature oocytes (n = 100) and different stages of embryos such as 2 cell (n = 50), 4 cell (n = 25), 8 cell (n = 12), 16 cell (n = 6), morula (n = 5) and blastocyst (n = 3). The total RNA isolated from the oocytes and embryos was reverse transcribed and subjected to real‐time polymerase chain reaction using sequence‐specific primers and SYBR green as the DNA dye. On sequence analysis, the nucleotide sequence of sheep FGF2 exhibited highest sequence similarity with cattle (100%) and least with rat and mouse (69.2%). At the deduced amino acid level, a highest degree of similarity was noticed with cattle, buffalo, goat, pig, camel and horse (100%) and lowest degree of identity with rat, human and mouse (98.2%). The FGF2 mRNA expression was higher in immature and mature oocytes and gradually decreases from 2‐cell stage of embryo to the blastocyst stage. More over a significant differences in FGF2 mRNA expression (p < .05) were observed between immature oocytes and all pre‐implantation stages of embryo. It can be concluded that FGF‐2 plays a significant role in pre‐implantation and early development of embryos in sheep.     

Isolation and Culture of Ovine and Bubaline Small and Large Pre-antral Follicles: Effect of Cyclicity and Presence of a Dominant Follicle

Studies were conducted to examine the effects of the cyclicity and the presence of a dominant follicle (DF) in ovary on the recovery and in vitro growth of pre-antral follicles (PFs) in sheep and buffalo. Small pre-antral follicles (SPFs, 100–250 μm) and large pre-antral follicles (LPFs, 250–450 μm) were isolated from slaughterhouse ovaries in the breeding seasons by a mechanical and enzymatic method. The sheep and buffalo PFs were cultured in vitro for 6 and 15 days, respectively, and examined for their growth, survival and antrum formation rates and growth rates of oocytes in cultured pre-antral follicles. The follicles of the sheep and buffalo were recovered and cultured simultaneously within replicates. The recovery rates (number per ovary) of both SPFs and LPFs were significantly (p < 0.05) higher in cyclic ewes (SPFs: 22.0 ± 3.3 vs 12.1 ± 2.6 and LPFs: 16.0 ± 3.6 vs 9.2 ± 1.8) and buffaloes (SPFs: 9.2 ± 1.3 vs 4.1 ± 1.0 and LPFs: 10.3 ± 2.7 vs 5.4 ± 0.7) compared with those recovered from acyclic ones. Presence of a DF in ovary significantly (p < 0.05) reduced the recovery rates of LPFs in ewes (9.06 ± 2.7 vs 16.4 ± 3.8) but had no effect in buffalo. Cyclicity of animals or follicular dominance had no effects on in vitro growth, survival and antrum formation rates and growth rates of oocytes in cultured PFs of SPFs and LPFs in both sheep and buffalo. The in vitro growth, survival and antrum formation rates of LPFs and growth rates of oocytes in cultured LPFs were significantly (p < 0.05) higher than those observed in SPFs in both sheep and buffalo. The overall recovery and growth rates of the PFs were lower in buffaloes compared with ewes.     

In vitro Development of Buffalo Oocytes in Media-containing Fluids from Different Size Class Follicles

Studies were conducted to investigate the effect of supplementation of fluid from different sized class [small (SFF, < 3 mm), medium (MFF, 3-8 mm) and large (LFF, > 8 mm)] of normal and cystic (CFF) ovarian follicles in oocyte culture media on oocyte maturation rate and embryo development in vitro and to test the efficacy of follicular fluid (FF) from different size classes as a whole oocyte maturation medium. Results suggested that FF were capable of developing buffalo oocytes to embryonic stage in vitro although its efficacy was lower than that of serum. Regardless of high maturation rates after in vitro maturation (IVM) in media containing FF or IVM in whole FF, low blastocyst rates were obtained after in vitro fertilization (IVF) and culture of embryos. Follicular fluid from small follicles had significantly (p < 0.05) higher potential of developing buffalo oocytes to embryonic stage in vitro than that from medium and large follicles. Cystic FF was not capable of supporting development of buffalo oocytes in vitro.