Fructooligosaccharide and Bacillus subtilis synbiotic combination promoted disease resistance, but not growth performance, is additive in fish.

Species diversification from major to minor carps for their sturdiness and initial higher growth, and also a quest for antibiotic-free aqua farming in the subcontinent, mandates search for and evaluation of alternatives. An experiment was performed to investigate the potential of fructooligosaccharide (FOS) and Bacillus subtilis (BS) (alone or as synbiotics) in promoting growth and immunity against infections in Labeo fimbriatus fingerlings. Six iso-nitrogenous and iso-lipidic diets containing combinations of two levels of FOS (0% and 0.5%) and three levels of BS (0, 104, 106 CFU/g feed) were fed to fish for 60 days. At the end of the feeding trial, twenty-four fish from each group were injected intra-peritoneally with pathogenic strain of Aeromonas hydrophila O:18 to test the immunoprotective efficacy of the supplements against bacterial infection. BS, but not FOS, significantly improved (P < 0.05) growth and feed utilisation attributes like percentage weight gain (PWG), specific growth rate (SGR) and feed conversion ratio (FCR). There were interactive effects of FOS and BS on PWG, SGR and FCR; however, the effects were not additive in nature. These beneficial effects of BS, alone or in combination with FOS, were corroborated by increased protease activity, microvilli density and diameter and number of goblet cells. Overall beneficial effects of FOS and BS included improved erythrocyte (RBC), hemoglobin (Hb), total protein and globulin levels. Total leucocyte (WBC) count and immunological parameters like respiratory burst activity of leucocytes (NBT reduction), lysozyme activity, albumin: globulin ratio and post-challenge survival were significantly improved by both FOS and BS, and their dietary combination yielded the highest improvement in these parameters. Synergistic effects of FOS and BS as dietary supplements indicate that a combination of 106 CFU/g BS and 0.5% FOS is optimal to improve growth, feed utilisation, immune functions, and disease resistance in L. fimbriatus fingerlings.

Monitoring and controlling ovarian activities in wild ungulates.

Reproductive success in females relies primarily upon better understanding of fundamental ovarian processes to facilitate captive reproductive management and ultimately the development of assisted reproductive technologies as an alternative tool for conservation breeding of endangered wildlife species. Wild ungulates (cervids and non-domestic bovids) are an extraordinarily diverse group of mammals with remarkable diversity in reproductive biology (anatomy, behaviour, physiology and seasonality). This indicates a clear need, and indeed a big challenge, for acquiring such basic reproductive knowledge in severely threatened ungulates before attempting even a simpler technology such as artificial insemination. Despite a few sporadic successes of artificial insemination technology in ungulates, there is much to learn, including optimizing estrus synchronization protocols and an animal-friendly fixed-time insemination procedure that would maximize breeding success with minimal stress to the animal. Recent advances in non-invasive methodologies for monitoring endocrine profiles and assessment of ovarian function in wildlife species enable us to understand and characterize basic reproductive events in a species. This review discusses non-invasive approaches being used for monitoring endocrine and ovarian activity in wild ungulates. Further, it reviews the effectiveness of different methodologies for control of estrus and ovulation in non-domestic bovids and cervids. Additionally, the challenges regarding their application in different ungulates as a standard routine practice like in livestock are addressed.

The Adequate Corpus Luteum: miR-96 Promotes Luteal Cell Survival and Progesterone Production.

Context: Inadequate progesterone production from the corpus luteum is associated with pregnancy loss. Data available in model species suggest important roles of microRNAs (miRNAs) in luteal development and maintenance. Objective: To comprehensively investigate the involvement of miRNAs during the ovarian follicle-luteal transition. Design: The effects of specific miRNAs on survival and steroid production by human luteinized granulosa cells (hLGCs) were tested using specific miRNA inhibitors. Candidate miRNAs were identified through microarray analyses of follicular and luteal tissues in a bovine model. Setting: An academic institution in the United Kingdom associated with a teaching hospital. hLGCs were obtained by standard transvaginal follicular-fluid aspiration from 35 women undergoing assisted conception. Intervention(s): Inhibition of candidate miRNAs in vitro. Main outcome measure(s): Levels of miRNAs, mRNAs, FOXO1 protein, apoptosis, and steroids were measured in tissues and/or cultured cells. Results: Two specific miRNA clusters, miR-183-96-182 and miR-212-132, were dramatically increased in luteal relative to follicular tissues. miR-96 and miR-132 were the most upregulated miRNAs within each cluster. Database analyses identified FOXO1 as a putative target of both these miRNAs. In cultured hLGCs, inhibition of miR-96 increased apoptosis and FOXO1 protein levels, and decreased progesterone production. These effects were prevented by small interfering RNA-mediated downregulation of FOXO1. In bovine luteal cells, miR-96 inhibition also led to increases in apoptosis and FOXO1 protein levels. Conclusions: miR-96 targets FOXO1 to regulate luteal development through effects on cell survival and steroid production. The miR-183-96-182 cluster could provide a novel target for the manipulation of luteal function.

Characterization of microRNAs differentially expressed during bovine follicle development.

Several different miRNAs have been proposed to regulate ovarian follicle function; however, very limited information exists on the spatiotemporal patterns of miRNA expression during follicle development. The objective of this study was to identify, using microarray, miRNA profiles associated with growth and regression of dominant-size follicles in the bovine monovular ovary and to characterize their spatiotemporal distribution during development. The follicles were collected from abattoir ovaries and classified as small (4-8  mm) or large (12-17  mm); the latter were further classified as healthy or atretic based on estradiol and CYP19A1 levels. Six pools of small follicles and individual large healthy (n=6) and large atretic (n=5) follicles were analyzed using Exiqon's miRCURY LNA microRNA Array 6th gen, followed by qPCR validation. A total of 17 and 57 sequences were differentially expressed (greater than or equal to twofold; P<0.05) between large healthy and each of small and large atretic follicles respectively. Bovine miRNAs confirmed to be upregulated in large healthy follicles relative to small follicles (bta-miR-144, bta-miR-202, bta-miR-451, bta-miR-652, and bta-miR-873) were further characterized. Three of these miRNAs (bta-miR-144, bta-miR-202, and bta-miR-873) were also downregulated in large atretic follicles relative to large healthy follicles. Within the follicle, these miRNAs were predominantly expressed in mural granulosa cells. Further, body-wide screening revealed that bta-miR-202, but not other miRNAs, was expressed exclusively in the gonads. Finally, a total of 1359 predicted targets of the five miRNAs enriched in large healthy follicles were identified, which mapped to signaling pathways involved in follicular cell proliferation, steroidogenesis, prevention of premature luteinization, and oocyte maturation.

Ejaculate characteristics, short-term semen storage and successful artificial insemination following synchronisation of oestrus in the Indian blackbuck antelope (Antilope cervicapra).

The blackbuck (Antilope cervicapra) is a small (20-30 kg) Indian antelope that is listed on Schedule I of the Indian Wildlife Protection Act, 1972. Studies were undertaken to develop assisted reproductive technologies, such as synchronisation of oestrus and non-surgical AI, to support the conservation and genetic management of this Indian antelope. Semen characteristics, testosterone levels and the feasibility of short-term cold storage of semen were investigated. Furthermore, different oestrous synchronisation protocols (norgestomet implants and prostaglandin injections) were evaluated for successful AI, defined as the birth of live young. Norgestomet ear implants and i.m. administration of pregnant mare's serum gonadotropin (PMSG) resulted in successful pregnancies in two of five inseminated females, but both had twin pregnancies that were delivered prematurely. In contrast, two injections of prostaglandin 11 days apart were effective in synchronising oestrus in the blackbuck. Transcervical AI in oestrous-synchronised animals 72 and 96 h after the second prostaglandin injection resulted in successful pregnancies in four of six inseminated females (67%) and resulted in the delivery of three live fawns. These studies demonstrate the potential application of AI technology for the conservation of endangered ungulates. To our knowledge, this is the first report regarding the synchronisation of oestrus and successful non-surgical AI in blackbuck.

Yohimbine antagonizes the anaesthetic effects of ketamine-xylazine in captive Indian wild felids.

OBJECTIVE: To determine the effectiveness of yohimbine as an antagonist of ketamine-xylazine anaesthesia in captive Asiatic lions (Panthera leo persica), tigers (Panthera tigris) and leopards (Panthera pardus). STUDY DESIGN: Prospective clinical trial. ANIMALS: Fifty-two healthy adult lions, 55 adult leopards and 16 adult male tigers. METHODS: Captive wild felids in Indian zoos were anaesthetized with a combination of ketamine (2.2-2.6 mg kg(-1)) and xylazine (1.1-1.3 mg kg(-1)) using a dart propelled from a blowpipe. Time to onset of anaesthesia, lateral recumbency and induction time were measured, and physiological variables (respiration, heart rate and rectal temperature) were recorded once after the onset of complete anaesthesia. Anaesthesia was antagonized at various time periods with an intravenous administration of either 0.1 or 0.15 mg kg(-1) yohimbine. Onset of arousal and time to complete anaesthetic recovery were recorded. RESULTS: A total of 123 immobilizations were conducted between 2000 and 2005. Anaesthetic induction was achieved in 15-25 minutes in all animals. Incidents of sudden recovery or life-threatening effects associated with immobilizations were not observed. Yohimbine effectively antagonized anaesthesia in all animals within 10 minutes without any excitatory behaviour compared to control animals. No adverse reactions/side effects to yohimbine were recorded except that a few leopards exhibited seizure-like signs for a short period immediately after yohimbine administration. The duration of anaesthesia had no significant effect on the recovery time in any of the animals. CONCLUSION AND CLINICAL RELEVANCE: Yohimbine antagonized the xylazine portion of ketamine-xylazine anaesthesia and thereby hastened recovery from anaesthesia in Asiatic lions, tigers and leopards.

Seasonal variations in semen characteristics, semen cryopreservation, estrus synchronization, and successful artificial insemination in the spotted deer (Axis axis).

Ten adult male spotted deer were monitored over a 2-year interval to determine seasonal variations in testicular size, semen characteristics and serum testosterone concentrations, and to determine if there was an association between season and type of antler. Mean (+/-S.E.M.) testicular volume (118.8+/-4.6 cm(3)), serum testosterone concentration (1.2+/-0.1 ng/mL), semen volume (4.1+/-0.6 mL), sperm concentration (338.3+/-24.9 x 10(6) mL(-1)), percentage of morphologically normal sperm (79.1+/-2.8%), and percentage of motile sperm (66.5+/-1.5%) were higher (P<0.05) in hard antler deer (peaked from March to May) than in deer with velvet antlers or in deer in which the antler has been shed. Thus, March-May was considered the physiologic breeding season for these deer; at this time, all stags had hard antlers. Furthermore, a Tris-citrate-based semen extender containing 4% glycerol and 20% egg-yolk was adequate for cryopreservation of semen. Estrus was induced with an implant containing norgestomet, timed transcervical AI was done with fresh semen, and 3 of 10 females were pregnant at 60 days, with fawns born 120 (premature), 240 and 243 days after AI. These results were considered a model for the use of assisted reproductive techniques to conserve other critically endangered deer species of India.

Anesthesia induced by administration of xylazine hydrochloride alone or in combination with ketamine hydrochloride and reversal by administration of yohimbine hydrochloride in captive Axis deer (Axis axis).

OBJECTIVE: To determine the anesthetic dose and cardiopulmonary effects of xylazine hydrochloride when used alone or in combination with ketamine hydrochloride and evaluate the efficacy of yohimbine hydrochloride to reverse anesthetic effects in captive Axis deer. ANIMALS: 35 adult (10 males and 25 females) Axis deer (Axis axis). PROCEDURES: All deer were anesthetized by IM administration of xylazine (3.5 mg/kg; experiment 1), a combination of ketamine and xylazine (1.25 and 1.5 mg/kg, respectively; experiment 2), or another combination of ketamine and xylazine (2.5 and 0.5 mg/kg, respectively; experiment 3). In addition, female deer were also anesthetized by IM administration of a third combination of ketamine and xylazine (1.5 and 1 mg/kg, respectively; experiment 4). Ten to 40 minutes after induction, anesthesia was reversed by IV administration of yohimbine (5, 8, or 10 mg). RESULTS: In male deer, experiment 3 yielded the most rapid induction of anesthesia. In females, experiment 4 yielded the best induction of anesthesia without adverse effects. All doses of yohimbine reversed anesthesia. Duration of anesthesia before administration of yohimbine had no effect on recovery time. CONCLUSIONS AND CLINICAL RELEVANCE: A combination of ketamine and xylazine can be used to induce anesthesia in Axis deer. Furthermore, anesthetic effects can be reversed by administration of yohimbine.

Estrus behavior and fecal steroid profiles in the Asiatic lion (Panthera leo persica) during natural and gonadotropin-induced estrus.

In this paper the behavior of the Asiatic lion was studied during natural and gonadotropin-induced estrus in relation to fecal estradiol and progesterone concentration. The average length of estrus was 5.4 days and no significant difference was observed between natural and gonadotropin-induced estrus. Vocalization and rolling were the major estrus behavioral activities of Asiatic lions and the frequency of these activities were similar in both natural and gonadotropin-induced estrus and treatment with exogenous gonadotropin did not alter estrus behavioral activities. A significant positive correlation was observed between fecal estradiol and frequency of estrus behavior during natural and gonadotropin-induced estrus. Following gonadotropin treatment estrus could be induced in 69% of animals and these induced animals ovulated following hCG treatment. This study reports for the first time the successful use of the non-invasive fecal steroid assay for monitoring the induction of estrus and ovulation in the Asiatic lion.

Semen characteristics, cryopreservation, and successful artificial insemination in the Blue rock pigeon (Columba livia).

The present study was undertaken in the Blue rock pigeon (Columba livia) to evaluate the annual semen characteristics, to identify a suitable extender for semen short-term storage, to determine a protocol for cryopreservation of semen and finally to check whether intracloacal insemination would lead to the birth of a chick. Semen characteristics such as semen volume, sperm concentration, sperm motility, and percentage of normal spermatozoa were maximum during the monsoon season. TALP was observed to be the most suitable semen extender and the sperm survived best at 37 degrees C at a dilution of 1:100 in TALP. Further, cryopreservation studies on pigeon semen indicated that 8% DMSO with or without egg yolk (20%) proved to be a better cryoprotectant compared to glycerol and polyethylene glycol. In addition, the slow freezing protocol was better than the fast-freezing protocol and about 40% of the cryopreserved spermatozoa were motile following thawing. Computer-aided semen analysis indicated that pigeon spermatozoa were extremely active immediately after dilution in TALP and exhibited linear trajectories persisting up to 9h. But, with time there was a time-dependent decrease in the velocity parameters (VAP, VSL, and VCL). Cryopreserved spermatozoa following thawing also exhibited linear trajectories but had reduced velocity as evident from the significant decrease in VAP, VSL, and VCL. Further, artificial inseminations using fresh semen resulted in 45% fertilization and birth of a live chick.