Jc Jacob

Temporal Relationships and Repeatability of Follicle Diameters and Hormone Concentrations within Individuals in Mares

Data were collected daily from 23 mares during two consecutive interovulatory intervals (IOIs). Several significant (p < 0.05) new observations on temporal relationships were made. The FSH increase that begins before ovulation temporarily plateaued on the day of discharge of follicular fluid into the peritoneal cavity in association with ovulation. During the declining portion of the pre-ovulatory oestradiol surge, an abrupt reduction in the rate of decrease occurred in synchrony with the peak of the LH surge and is consistent with a negative effect of LH on oestradiol. Repeatability within mares was based on the following positive and significant correlations between the two IOIs: (i) length of the interval between ovulations and between ovulation and the beginning of follicle deviation; (ii) diameter of the pre-ovulatory follicle on days -3 to -1; (iii) number of follicles in diameter classes of 2-5 mm (correlation for 22/23 days of the IOI), 5.1-10 mm (18/23 days), 10.1-15 mm (12/23 days) and 15.1-20 mm (12/23 days) and (iv) concentrations of FSH (18/23 days) and LH (22/23 days). The greatest repeatability for the follicle-diameter classes occurred in the 2-5 mm class, and thereafter the repeatability progressively decreased as the diameters for the classes increased. Results demonstrated measurable repeatability within mares for several end points between consecutive IOIs.

Effects of age on follicle and hormone dynamics during the oestrous cycle in mares

The effects of age (young: 5-6 years; intermediate: 10-14 years; old: > or =18 years) on follicle and hormone dynamics during an interovulatory interval (IOI; n = 46) and on preovulatory oocytes and concentrations of follicular fluid factors (n = 44) were studied in mares. Old mares were not approaching senescence, as indicated by regular lengths of the IOI (19-27 days) during the period May-October. The IOI was 1 day longer (P < 0.05) in the old group than in the two younger groups and was associated with a slower (P < 0.05) growth rate of the ovulatory follicle. The old group had diminished follicle activity, as indicated by significantly smaller and fewer follicles. Concentrations of FSH did not differ among age groups, except that the maximum concentration was greater (P < 0.05) in the old group. Concentrations of LH were greater (age x day interaction; P < 0.03) in the young group throughout the ovulatory LH surge and may have played a role in a shorter (P < 0.05) interval from maximum diameter of the preovulatory follicle to ovulation. Maximum circulating concentrations of oestradiol during the preovulatory surge were greatest (P < 0.05) in the young group. No effects of age were detected on oocyte morphology. Concentrations of ovarian steroids in preovulatory follicular fluid were not affected by the age of the mares, but concentrations of free insulin-like growth factor-1 were greater (P < 0.05) in the old group. The results indicate the importance of considering the potential confounding effects of age in experimental protocols and for considering age in the development of theriogenology programmes.

Age-related dynamics of follicles and hormones during an induced ovulatory follicular wave in mares

An ovulatory follicular wave was induced by ablation of follicles > or =6mm and treatment with prostaglandin F2alpha (PGF) on Day 10 (ovulation=Day 0). Follicle and hormone dynamics of the induced waves were compared among three age groups: young (5-6 y, n=14 waves), intermediate (10-14 y, n=16), and old (> or =18, n=15). During the common-growth phase of the induced wave (Days 12-17), diameter of the future ovulatory follicle was not different among ages, but the young group had more (P<0.05) follicles that reached > or =10mm. The number was correlated (r=+0.7; P<0.0001) within mares between consecutive interovulatory intervals, indicating repeatability. Concentrations of LH increased in all age groups during Days 12-17, but were greatest (P<0.002) in the young group and continued to be greater (P<0.0001) throughout the ovulatory LH surge. During several days before Day-1, there were no age-related effects on systemic estradiol concentrations, diameter of the preovulatory follicle, or B-mode echotexture or color-Doppler signals of blood flow in the follicle wall. Interpretations were: (1) greater number of follicles in the young group reflected a greater follicle reserve, (2) greater LH concentrations throughout the ovulatory surge in the young group reflected a more positive response to an extraovarian/environmental influence after removal of the negative effect of progesterone, and (3) lower LH concentrations in the older groups were adequate for the preovulatory changes in the follicle.

Follicle and systemic hormone interrelationships during spontaneous and ablation-induced ovulatory waves in mares

The characteristics of ovulatory follicular waves were studied for spontaneous waves and waves induced during the next estrous cycle by ovarian follicle ablations and administration of PGF2alpha 10 days after ovulation in 21 mares. In the induced group, both the days of the FSH surge and day of deviation were more synchronized, LH concentrations were greater before and after deviation, estradiol concentrations were greater after deviation, and the ovulatory follicle grew at a faster rate (3.4+/-0.2 compared with 2.7+/-0.1 mm/day). The frequency of two dominant follicles/wave was not different between induced waves (7 of 21) and spontaneous waves (9 of 21), but both dominant follicles ovulated more frequently in induced waves (6 of 7 waves compared with 0 of 9).

Follicle Deviation in Ovulatory Follicular Waves with One or Two Dominant Follicles in Mares

The follicle and hormone aspects of diameter deviation and development of one dominant (>/=28 mm) follicle (1DF) vs two dominant follicles (2DF) were studied in 32 ovulatory follicular waves in mares. Follicles were ranked each day as F1 (largest) to F3. The beginning of deviation was designated day 0 and preceded the first increase in the differences in diameter between F1 and F2 in the 1DF group and between a combination of F1 and F2 vs F3 in the 2DF group. One dominant follicle and 2DF developed in 21 (66%) and 11 (34%) waves, respectively. Double ovulations occurred in only one of the waves with 2DF. In 8/11 waves with 2DF, a second deviation occurred between F1 and F2 on 2.5 +/- 0.4 days after the first deviation. On day 0, 1DF and 2DF waves were similar in number of days after ovulation, number of follicles, difference in diameter between F1 and F2, and plasma concentrations of LH, estradiol and immunoreactive inhibin. The interval from maximum FSH concentration to day 0 was longer (p < 0.05) and FSH concentration was lower (p < 0.05) on days -1 to 4 in the 2DF group. The similarities on day 0 in the characteristics of 1DF and 2DF waves despite the differences in the declining portions of the FSH profile indicated that a specific day of the FSH decline or a specific concentration were not factors in initiating deviation. Unlike reported results in heifers, the results in mares did not indicate a hormonal basis for the development of 2DF or two deviations.

Induction of haemorrhagic anovulatory follicles in mares

A follicular wave and luteolysis were induced in mares by ablation of follicles > or =6 mm and treatment with prostaglandin F(2alpha) (PGF) on Day 10 (where ovulation = Day 0). The incidence of haemorrhagic anovulatory follicles (HAFs) in the induced waves (20%) was greater (P < 0.007) than in preceding spontaneous waves (2%). Hormone and follicle dynamics were compared between induced follicular waves that ended in ovulations (ovulating group; n = 36) v. HAFs (HAF group; n = 9). The day of the first ovulation or the beginning of HAF formation at the end of an induced wave was designated as post-treatment Day 0. The mean 13-day interval from Day 10 (PGF and ablation) to the post-treatment ovulation was normalised into Days 10 to 16, followed by Day -6 to Day 0 relative to the post-treatment ovulation. Concentrations of LH were greater (P < 0.05) in the HAF group than in the ovulating group on Days 10, 11, 12, 14, -3 and -2. The HAF group had greater (P < 0.003) LH concentrations on Day 10 of the preceding oestrous cycle with spontaneous ovulatory waves. The diameter of the largest follicle was less (P < 0.05) in the HAF group on most days between Day 13 and Day -1 and this was attributable to later (P < 0.002) emergence of the future largest follicle at 6 mm in the HAF group (Day 12.4 +/- 0.5) than in the ovulating group (Day 11.3 +/- 0.1). The results indicate that the high incidence of HAFs after PGF and ablation was associated with later follicle emergence and immediate and continuing greater LH concentration after PGF treatment, apparently augmented by an inherently high pretreatment LH concentration.

Effect of embryo age and recipient asynchrony on pregnancy rates in a commercial equine embryo transfer program

In the present study, 809 uterine flushes and 454 embryo transfers performed in mares over a 4-yr interval were examined to evaluate the effects of: (1) the day of embryo collection on recovery rates; (2) the degree of synchrony between donor and recipient mares on pregnancy rates; (3) the recipient day post ovulation on pregnancy rates; and (4) the age of the embryo at recovery on pregnancy rates at 60 days. Uterine flushes were performed on Days 6, 7, 8, 9, and 10 (Day 0 = ovulation) and embryos were transferred to recipients with degrees of synchrony varying between +1 to -6 (recipient ovulated 1 day before through 6 days after the donor). Recipient mares ranged from 2 to 8 days post ovulation. Embryo recovery rates were similar for flushes performed on Day 7 (61%), Day 8 (66%), Day 9 (59%), and Day 10 (56%), but the embryo recovery rate was lower (P < 0.03) for flushes performed on Day 6 (42%) compared with all other days. Pregnancy rates for various degrees of synchrony were as follows: +1 (71%), 0 (77%), -1 (68%), -2 (63%), -3 (66%), -4 (76%), -5 (61%), and -6 (27%). The -6 day of degree of synchrony had the lowest (P < 0.05) pregnancy rate compared with all other days, but there was no significant difference among +1 to -5 days. There was a lower (P < 0.05) pregnancy rate for embryos transferred to recipient mares on Day 2 (33%) compared with mares on Day 3 (66%), Day 4 (66%), Day 5 (62%), Day 6 (55%), Day 7 (58%), and Day 8 (56%). Pregnancy rate was higher (P < 0.05) for Day 7 (76%) embryos compared with Day 6 (50%), Day 8 (64%), and Day 9 (44%) embryos; Day 9 embryos resulted in lower (P < 0.05) pregnancy rates than Days 7 or 8 embryos. In conclusion, this study demonstrated that: (1) embryo recovery rates between Days 7 and 10 were similar and acceptable (e.g., 63% 488/771); (2) the degree of synchrony between donor and recipient mares does not need to be as restricted as previously reported in horses. Acceptable pregnancy rates (e.g., 70%, 99/142) were obtained even when recipient mares ovulated 4 to 5 days after the donors; (3) similar pregnancy rates were obtained when recipient mares received embryos within a large range of days post ovulation (Days 3 to 8); and (4) Day 7 embryos produced higher pregnancy rates when compared with Days 8 and 9 embryos. In clinical terms, the application of these new findings will be beneficial to large equine embryo transfer operations in producing more pregnancies per season.

Development of one vs multiple ovulatory follicles and associated systemic hormone concentrations in mares.

Ablation of follicles > or = 6 mm in diameter and treatment with PGF2alpha 10 days after ovulation were used to induce the development of ovulatory waves. Comparisons were made between induced waves with one (33 waves, 72%) and multiple (13 waves, 28%) ovulatory follicles. Diameter deviation was defined as the separation of follicles into dominant and subordinate categories. Multiple ovulatory follicles were preceded by more (p < 0.001) follicles > or = 20 mm at the beginning of deviation, higher LH preceding deviation (approached significance, p < 0.08), lower (p < 0.05) concentrations of FSH on the day of deviation and thereafter, and higher (p < 0.0003) oestradiol by 2 days after deviation. During the peri-ovulatory period, systemic hormone concentrations for waves with multiple ovulations involved higher oestradiol before ovulation (approached significance, p < 0.07), lower FSH (p < 0.04) before and after ovulation, and both higher progesterone (p < 0.05) and lower LH (p < 0.05) beginning the day after ovulation. Results indicated that by the beginning of deviation there were more follicles > or = 20 mm and subsequently greater oestradiol production in waves that led to the development of multiple ovulatory follicles, and during the peri-ovulatory period differences between one and multiple ovulations were consistent with the negative effects of the ovarian hormones on the gonadotropins.

Prenatal Development of the Digestive System in the Horse

Since the horse has a highly precocial reproductive strategy, most organs are functionally well developed at birth and thus, embryonic and fetal life is interesting. Data on the development of important organs are very limited. Here, we detailed macroscopically and histologically the equine digestive system, focusing on the first third of gestation. At 21 days, the oral cavity was an empty space, and the liver contained proliferating endodermal cells. At 25 days, a fusiform stomach and the pancreatic bud were present. At 28 days, a small tongue and the esophagus occurred. At 30 days, primary and secondary palates were developed, the liver contained cords of hepatocytes, and the pancreas was triangular. At 40 days, crypts had formed in the intestinal loops, cell differentiation was observed in the hepatic parenchyma, and the pancreas was elongated. Pancreatic acini and islets were observed in fetuses of 50 days and intestines were highly convoluted. Three segments of the pharynx were distinguishable at 75 days. At 105 days, the intestinal villi were wide with round tips; especially, the liver, stomach, and oral cavity showed key steps of anatomical and cellular differentiation in early fetuses, whereas other areas, such as pancreas or pharynx were still immature in the investigated phase. Pluripotency analysis using Oct4 showed initial intense staining in all of the digestive system tissues and a later decreased becoming restricted to specific cell layers. In conclusion, our data may contribute to perform a chronological reference of developmental events for approaches predicting pregnancy disorders in horses. Anat Rec, 297:1218–1227, 2014.

Organogenesis of the Musculoskeletal System in Horse Embryos and Early Fetuses.

Musculoskeletal system development involves heterotypical inductive interactions between tendons, muscles, and cartilage and knowledge on organogenesis is required for clarification of its function. The aim of this study was to describe the organogenesis of horse musculoskeletal system between 21 and 105 days of gestation, using detailed macroscopic and histological analyses focusing on essential developmental steps. At day 21 of gestation the skin was translucid, but epithelial condensation and fibrocartilaginous tissues were observed on day 25 of pregnancy. Smooth muscle was seen in lymphatic and blood vessel walls and the beginning of cartilaginous chondrocranium was detected at day 30 of gestation. At day 45, typical chondroblasts and chondrocytes were observed and at day 55, mandibular processes expanded toward the ventral midline of the pharynx. At day 75, muscles became thicker and muscle fibers were seen developing in carpal and metacarpal joints with the beginning of the ossification process. At day 105, major muscle groups, similar to those seen in an adult equine, were observed. The caudal area of the nasal capsule and trabecular cartilages increased in size and became ossified, developing into the ethmoid bone. The presence of nasal, frontal, parietal, and occipital bones was observed. In conclusion, novel features of equine musculoskeletal system development have been described here and each process was linked with an early musculoskeletal event. Data presented herein will facilitate a better understanding of the equine muscular system organogenesis and aid in the detection of congenital deformities. Anat Rec, 299:722–729, 2016.