Evolution of pig intestinal stem cells from birth to weaning.

Pig intestinal epithelium undergoes a complete renewal every 2 to 3 days that is driven by intestinal stem cells (ISCs) located at the crypt base in their niche. Intestinal stem cells generate a pool of highly proliferative transit-amplifying cells, which either migrate up the villus and differentiate into enterocytes and secretory cells or migrate towards the base of the crypt where they differentiate into Paneth cells that secrete antimicrobial peptides. The balance between ISCs' self-renewal and differentiation controls intestinal epithelial homeostasis; therefore, ISCs are essential for ensuring intestinal epithelial integrity. Detailed knowledge of these mechanisms in pig and other domestic species is very limited. Therefore, the aim of this work was to characterize ISC from birth to weaning. We analysed the duodenum, jejunum and colon of six piglets at birth, 6-day-old nursing piglets and 28-day-old weanlings, one week after weaning. We immunolocalized homeobox only protein+ (HOPX) and sex-determining region Y-box 9+ (SOX9) cells that identify quiescent and active ISC, respectively. The volume of ISCs was quantified with stereological methods and was compared to that of mitotic cells expressing proliferating cell nuclear antigen and apoptotic cells identified by the presence of cleaved caspase-3. Furthermore, we compared all these values with crypts and villi measurements and their ratio. Our results indicated that both quiescent and active ISCs are present in pig intestine from birth to weaning and are localized in the crypts of the small and large intestine. However, both markers were also observed along the villi and on the colon luminal epithelium, suggesting that at these stages, pig mucosa is still immature. Weaning induced a dramatic reduction of both HOPX+ and SOX9+ cells, but SOX9+ cells underwent a significantly greater reduction in the small intestine than in the colon. This suggests that the two ISC types are differentially regulated along the intestinal tracts. Overall, the pig ISC complex has many similarities with its murine counterpart, but also has some differences. These include active ISC not showing the typical columnar base morphology as well as the absence of bona fide Paneth cells. This is the first description of ISC dynamics during pig's early life and provides useful reference data for future studies, aimed at targeting ISC for the development of efficient alternatives to in-feed antibiotics for preserving intestinal integrity.

Effect of nutritional state on semen characteristics, testicular size and serum testosterone concentration in Sanjabi ram lambs during the natural breeding season.

This experiment was conducted to investigate the effect of plane of nutrition on body weight, average daily gain, dry mater intake, semen characteristics, serum testosterone concentration and testicular circumference of Sanjabi ram lambs during the natural breeding season. Sanjabi ram lambs (n = 20) that were 8 months of age were penned under natural photoperiod at latitude 34°18'N for a period of 9 months. The control group (C, n = 10) received a diet consisting of 80% alfalfa and 20% concentrate, providing 2.18 Mcal metabolizable energy and 130.0 g /kg DM crude protein, whereas, treatment group (T, n = 10) was fed with 65% alfalfa and 35% concentrate, providing 2.34 Mcal metabolizable energy and 160.0 g/kg DM crude protein. Body weight, additive daily gain and feed intake in T group were significantly greater than those obtained in C group. Body weight and testicular circumference increased at a steady rate throughout the experiment. All semen variables (except percentage of abnormal sperm and semen pH), serum testosterone concentration and testicular circumference were positively influenced by nutritional state (P < 0.05). Interaction of nutritional state with season was found for semen volume, sperm concentration and abnormal sperm, but there was no interaction on the total sperm, progressive motility, live sperm, semen pH and semen index. It is concluded that the reproductive activity of growing Sanjabi ram lambs is affected by nutritional state. These results also demonstrated a monthly pattern in reproductive characteristics of Sanjabi ram lambs, independent of the nutritional state.

Comparison of endocrine changes, timing of ovulations, ovarian follicular growth, and efficacy associated with Estradoublesynch and Heatsynch protocols in Murrah buffalo cows (Bubalus bubalis).

Poor estrus expression and the difficulty encountered in predicting the time of ovulation compromise the reproductive efficiency of Murrah buffalo cows. Synchronization of ovulation and timed artificial insemination are able to precisely control the time of ovulation and thus avoid the need for estrus detection. Recently, the Estradoublesynch protocol (administration of a PGF2α injection 2 days before Heatsynch protocol; GnRH 0, PGF2α 7, estradiol benzoate [EB] 8) was developed that precisely synchronized ovulation twice, i.e., after GnRH and EB injections and resulted in satisfactory pregnancy rates in Murrah buffaloes. The present study was conducted on 104 cycling and 31 anestrus buffaloes to compare (1) the endocrine changes, timing of ovulations, ovarian follicular growth, and efficacy of Estradoublesynch and Heatsynch protocols in cycling and (2) the efficacy of Estradoublesynch and Heatsynch protocols for the improvement of fertility in cycling and anestrus Murrah buffalo cows. Ovulation was confirmed after all GnRH and EB treatments by ultrasonographic examination at 2-hour intervals. Plasma progesterone and total estrogen concentrations were determined in blood samples collected at daily intervals, beginning 2 days before the onset of protocols until the day of second ovulation detection. Ovulatory follicle size was measured by ultrasonography at six time points (first PGF2α administration of Estradoublesynch protocol every 2 days before the onset of Heatsynch protocol, GnRH administration of both protocols, 2 hours before ovulation detection after GnRH administration of both protocols, second PGF2α injection of Estradoublesynch protocol, PGF2α injection of Heatsynch protocol, EB injection of both protocols and, 2 hours before ovulation detection after EB administration of both protocols). Plasma LH, total estrogen, and progesterone concentrations were determined in blood samples collected at 30-minute intervals for 8 hours, beginning GnRH and EB injections, and thereafter at 2-hour intervals until 2 hours after the detection of ovulation. The first ovulatory rate was significantly higher (P<0.05) in the Estradoublesynch protocol (84.6%) than that in the Heatsynch protocol (36.4%). The first LH peak concentration (74.6±10.4 ng/mL) in the Estradoublesynch protocol was significantly higher (P<0.05) than that of the Heatsynch protocol (55.3±7.4 ng/mL). In Estradoublesynch protocol, the total estrogen concentration gradually increased from the day of GnRH administration coinciding with LH peak, and then gradually declined to the basal level until the time of ovulation detection. However, in Heatsynch protocol, the gradual increase in total estrogen concentration after GnRH administration was observed only in those buffalo cows, which responded to treatment with ovulation. In both Estradoublesynch and Heatsynch protocols, ovulatory follicle size increased by treatment with GnRH and EB until the detection of ovulation. The pregnancy rate after the Estradoublesynch protocol (60.0%) was significantly higher (P<0.05) than that achieved after the Heatsynch protocol (32.5%). Satisfactory success rate using the Estradoublesynch protocol was attributed to the higher release of LH after treatment with GnRH, leading to ovulation in most of the animals and hence creating the optimum follicular size at EB injection for ovulation and pregnancy to occur.

Endocrine changes, timing of ovulation, ovarian follicular growth and efficacy of a novel protocol (Estradoublesynch) for synchronization of ovulation and timed artificial insemination in Murrah buffaloes (Bubalus bubalis).

Experiments were conducted to investigate (1) ovarian follicular growth, timing of ovulation and associated endocrine changes (progesterone, estrogen, and LH) in cycling, and (2) efficacy in terms of pregnancy rate in cycling and anestrus Murrah buffaloes subjected to the Estradoublesynch protocol (prostaglandin F2α [PGF2α] 0, GnRH 2, PGF2α 9, estradiol benzoate, EB 10). Twelve cycling buffaloes were subjected to the Estradoublesynch protocol and observed for ovulation, follicle size, and endocrine changes after EB treatment. Ovulation occurred in 12 of 12 buffaloes (100%) at 48.5 ± 1.6 hours (range, 38.0-56.0) after EB treatment. Plasma LH, total estrogen, and progesterone concentrations were determined in intensive blood samples collected after EB treatment. Peak LH concentration of 34.2 ± 7.7 ng/mL (range, 17.8-178.5) occurred at 18.3 ± 0.8 hours (range, 14.0-22.0) after EB treatment. Peak total estrogen of 50.8 ± 6.9 pg/mL (range, 32.3-82.7) occurred 5.7 ± 1.0 hours (range, 2.0-14.0) after EB treatment. Follicle size was gradually increased from second PGF2α injection (9.7 ± 0.3 mm; range, 8.0-12.0) until ovulation was detected (12.9 ± 0.4 mm; range, 11.0-15.0). Fourteen cycling and 11 anestrus buffaloes were subjected to the Estradoublesynch protocol, with timed artificial insemination (TAI) 48 and 60 hours after EB treatment, and 58 cycling buffaloes were inseminated after spontaneous estrus (control group). Pregnancy rates were 62% for TAI of cycling buffaloes, 64% for anestrus buffaloes, and 34.5% for control group. Our observations demonstrated that the Estradoublesynch protocol followed by TAI satisfactory enhanced pregnancy rates in both cycling and anestrus buffaloes.

The endocrine changes, the timing of ovulation and the efficacy of the Doublesynch protocol in the Murrah buffalo (Bubalus bubalis).

Experiments were conducted to investigate (a) the timing of ovulation and the associated endocrine changes (progesterone, estrogen and LH) during estrous cycle and (b) the efficacy, with respect to the pregnancy rate, in cycling and anestrus in Murrah buffaloes subjected to the Doublesynch protocol during the low breeding season. In experiment 1, 10 cycling buffaloes were administered PGF(2α) on day 0 (without regard to the estrous cycle stage), GnRH on day 2, a second PGF(2α) injection on day 9, and a second GnRH injection on day 11. Transrectal palpation was performed at 2-h intervals after the first and second GnRH treatments until ovulation was detected or for upto 96 h. The plasma progesterone and total estrogen concentrations were determined in blood samples collected at daily intervals starting 2 days before the onset of the protocol and continued until the day of the second detected ovulation. The plasma LH and total estrogen concentrations were measured in blood samples collected at 30-min intervals for 8h following the first and second GnRH injections and thereafter at 2-h intervals until 2h after the detection of ovulation. Ovulation occurred in 9/10 buffaloes (90%) at 22.2 ± 1.2 h (mean ± S.E.M.; range 18.0-26.0 h) and 10/10 buffaloes (100%) at 23.2 ± 1.0 h (mean ± S.E.M.; range 20.0-28.0 h) after the first and second GnRH treatments, respectively. The peak LH concentrations of 99.8 ± 28.5 ng/ml (range 37.8-320.0 ng/ml) and 62.3 ± 11.9 ng/ml (range 20.9-143.9 ng/ml) occurred 2.1 ± 0.3 h (range 1.0-3.5 h) and 2.3 ± 0.3 h (range 0.5-3.0 h) after the first and second GnRH treatments, respectively. The total estrogen concentration gradually increased from the day of both the first and second PGF(2α) administrations until the LH peak (with great variability) and then gradually declined to the basal level, which was reached at the time ovulation was detected. In experiment 2, 10 cycling and 11 non-lactating anestrus buffaloes were subjected to the Doublesynch protocol with timed artificial insemination (TAI) 16 and 24 h after the second GnRH treatment, and 55 cycling buffaloes were inseminated after spontaneous estrus was detected (control group). The pregnancy rates were 60% using TAI on cycling buffaloes (experiments 1 and 2), 55% for anestrus buffaloes (experiment 2), and 27.3% for cycling buffaloes inseminated following spontaneous estrus. The overall pregnancy success rates after the Doublesynch protocol in both cycling and anestrus buffaloes increased by 30.8% compared to spontaneous estrus (58.1% vs. 27.3%). In conclusion, the Doublesynch protocol effectively synchronized ovulation twice (after the first and second GnRH treatments) irrespective of the stage of estrous cycle in Murrah buffaloes. The study also demonstrated that the Doublesynch protocol followed by TAI significantly (P<0.005) enhanced the pregnancy rate in cycling and anestrus buffaloes in comparison to untreated controls during the low breeding season.