The Effects of Cryopreservation on the Morphometric Dimensions of Iberian Red Deer (Cervus elaphus hispanicus) Epididymal Sperm Heads

Computer-automated sperm-head morphometry was used in this study to determine the effects of cryopreservation on red deer sperm-head morphometry. Epididymal sperm samples were collected from 40 mature stags and were divided. One portion was diluted at room temperature in a Tris-citrate egg yolk medium, containing 6% glycerol. A microscope slide was prepared from single extended sperm samples prior to freezing. The remainder of each sample was frozen in nitrogen vapours. After thawing, sperm smears were prepared as described above. All slides were air dried and stained with Hemacolor. The sperm-head dimensions for length, width, area, perimeter and shape factor (length/width), for a minimum of 135 spermatozoa were determined for each slide by means of the Sperm-Class Analyser (SCA). Firstly, our results show that cryopreservation substantially reduced (p < 0.001) sperm motility and plasma membrane and acrosome integrities. In addition, sperm heads were significantly smaller in cryopreserved spermatozoa than in the companion extended samples for area (32.05 microm2 vs 32.56 microm2; p < 0.05), length (8.46 microm vs 8.53 microm; p < 0.0001) and shape factor (1.833 vs 1.849; p < 0.0001) for all stags. These differences were found within 29 of 40 stags (75%) for at least three of the morphometric parameters. The individual variability (CV) of sperm head measurements from extended samples was negatively correlated (p < 0.005) with the per cent of change in sperm head measurements after cryopreservation for area (r = -0.465), width (r = -0.483) and perimeter (r = -0.375). Thus, the lower the sperm head variability in the extended samples, the greater the sperm change as a consequence of the cryopreservation. These results suggest that the variability (heterogeneity) in sperm head dimensions of individual stags may be a good indicator of sperm freezability.     

Luteal activity and effect of dietary energy restriction on follicular development in lactating cows

The aim of this research has been to evaluate the presence of anomalies in the ovarian cycle activity during postpartum and to verify whether 72‐hr dietary fasting during the dominance phase, the phase before ovulation, might modify the ovarian follicle population. The presence of anomalies in ovarian cycle activity has been evaluated in 30 Italian Friesian cows starting from 20 days postpartum until 211 days of lactation. Long oestrus and brief dioestrus or scarce luteal activity have been the main anomalies found through measuring progesterone concentrations in the whey. Until 100 days of lactation, the BCS values of the problematic animals have been significantly lower than those in animals with normal ovarian activity. After 100 days of lactation, the ovarian anomalies continued to appear despite the fact that all the animals have reached comparable BCS values. Starting from the results of this trial, the effect of 72‐hr dietary fasting on dominant follicles has been studied in six cows. Ultrasonography revealed that the diameter of the follicles at 71 days postpartum has been significantly lower than at 181 days. A 72‐hr dietary restriction at 101 and 211 days postpartum did not affect the size of the dominant follicle. However, at 101 days postpartum, half of the animals presented follicular cysts. The effect of fasting differed if the animal has been in early postpartum or 211 days of lactation. Further researches are necessary to understand how different metabolic conditions can modify the follicular population but on the other hand the study shows the utility for farmers and field veterinarians of monitoring the resumption of the ovarian cycle postpartum through the whey progesterone concentrations.     

Uterine Infection Influences Size and Follicular Fluid Composition of the Largest Follicle in Buffalo (Bubalus bubalis)

The effect of uterine infection on size and follicular fluid composition of the largest follicle was studied in buffalo. Reproductive tracts were collected from 102 graded Murrah buffaloes at an abattoir. Uterine infection was diagnosed by physical examination of uterine mucus, white side test and uterine cytology. Samples with pus‐containing mucus, positive reaction on white side test and/or >5% neutrophils were considered to be positive for uterine infection. Diameter of the largest follicle was measured, and follicular fluid was aspirated and assayed for nitric oxide (NO), ascorbic acid (AA), cholesterol, oestradiol (E₂) and progesterone (P₄). Infected buffaloes had smaller‐sized (p < 0.0001) largest follicles than non‐infected buffaloes. Follicular fluid collected from the largest follicle in infected buffaloes had greater (p < 0.0001) NO and P₄ concentrations coincident with lesser AA (p < 0.001), cholesterol (p < 0.0001) and E₂ (p < 0.0001) concentrations. Results indicated that uterine infection has an inhibitory effect on growth of the largest follicle in buffalo. The changes in follicular fluid composition in infected buffaloes suggest that the direct effect of uterine infection on ovarian function may be mediated through an alteration in the follicular microenvironment. Greater NO and lesser AA concentrations in the follicular fluid of infected animals are novel findings.     

Effect of Suppression of FSH with a GnRH Antagonist (Acyline) Before and During Follicle Deviation in the Mare

A GnRH antagonist (Acyline) was used to study the role of FSH in early development of a follicular wave in 61 mares. In Experiment 1, a single dose of 3 mg per mare, compared with 0 and 1 mg, suppressed both the FSH and follicle responses to exogenous GnRH. In Experiment 2, high concentrations of FSH were induced by two successive ablations of all follicles ≥ 6 mm on days 10 and 13 (day 0 = ovulation). A single treatment with Acyline resulted in significantly greater suppression of plasma concentrations of FSH than a single treatment with charcoal-extracted follicular fluid (source of inhibin) or oestradiol. Suppression of FSH was not significantly different between the group treated with Acyline alone and a group treated with a combination of Acyline, inhibin and oestradiol. In Experiment 3, all follicles were ablated on day 10 to induce an FSH surge and a new follicular wave. Acyline treatment on day 10 resulted in an immediate decrease in FSH, without a significant effect on day of emergence of a new wave or growth of follicles from 7 to 11 mm on days 11–13. Treatment on day 15, a day before expected follicle deviation and after the peak of the wave-stimulating FSH surge, resulted in an immediate decrease in FSH and cessation of follicle growth. Results indicated that growth of follicles for about 2 days after wave emergence was independent of FSH. In contrast, during the decline in the wave-stimulating FSH surge and before follicle deviation, growth of follicles was dependent on FSH.     

hCG‐Induced Ovulation in Thoroughbred Mares Does Not Affect Corpus luteum Development and Function During Early Pregnancy

Our aim was to compare Corpus luteum (CL) development and blood plasma concentration of progesterone ([P₄]) in thoroughbred mares after spontaneous (Control: C) or human chorionic gonadotrophin (hCG)‐induced ovulation. Lactating mares (C = 12; hCG = 21) were daily teased and mated during second oestrus post‐partum. Treated mares received 2500 IU hCG i.v. at first day of behavioural oestrus when dominant follicular size was >35, ≤42 mm and mated 12–24 h after. Control mares in oestrus were mated with dominant follicular size ≥45 mm. Dominant follicle before ovulation, CL and gestational sac were measured by ultrasound and [P₄] by radioimmunoassay (RIA). Blood sampling and ultrasound CL exams were done at days 1, 2, 3, 4, 8, 12, 16, 20, 25, 30, 35, 40, 45, 60 and 90 after ovulation and gestational sac from day 12 after ovulation in pregnant (P) mares; non‐pregnant (NP) were followed until oestrus returned. Data analyses considered four subgroups: hCG‐P, hCG‐NP, C‐P and C‐NP. Preovulatory follicular size was smaller in hCG mares than in C: 39.2 ± 2.7 mm vs 51.0 ± 1.8 mm (p < 0.0001). All hCG mares ovulated 24–48 h after treatment and presented similar oestrus duration as controls. C. luteum size in P mares showed the same pattern of development through days 4–35, presenting erratic differences during initial establishment. Thus, on days 1 and 3, CL was smaller in hCG‐P (p < 0.05); while in hCG‐NP, CL size was greater than in C‐NP on day three (p = 0.03). Corpus luteum size remained stable until day 90 in hCG‐P mares, while in C‐P a transient and apparently not functional increase was detected on days 40 and 45 (p < 0.05) and the decrease from day 60 onwards, made this difference to disappear. No differences were observed in [P₄] pattern between P, or between NP subgroups, respectively. So, hCG‐induced ovulation does not affect CL development, neither [P₄] during early pregnancy. One cycle pregnancy rate tended to be lower in hCG mares while season pregnancy rates were similar to controls.