[Peroneal nerve palsy in anorexia nervosa: three cases].
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Adolescent↗
Biomedical subjects
J F Roche
Publications and source records attributed to J F Roche.
Identification of potential intrafollicular factors involved in selection of dominant follicles in heifers.
A surgical procedure to aspirate follicular fluid concurrently from individual follicles from the same heifer was validated and used to determine if intrafollicular amounts of estradiol, progesterone, inhibins, activin-A, follistatins, and insulin-like growth factor binding proteins (IGFBP) differed for the future dominant compared with subordinate follicles during selection of the first wave dominant follicle. Heifers were subjected to surgery and aspiration of follicular fluid from the two or three largest follicles on Day 3 of the estrous cycle (approximately 1.5 days after emergence). Ultrasound was used to determine the fate of each aspirated follicle after surgery. At aspiration, diameter of the future dominant and largest subordinate follicle was similar in heifers. However, estradiol was higher, whereas IGFBP-4 was lower in the future dominant compared with the largest or next largest subordinate follicles. Also, the future dominant follicle in most cohorts had the highest estradiol and lowest IGFBP-4 compared with future subordinate follicles. We concluded that: IGFBP-4 and estradiol may have key roles in determining the physiological fate of follicles during selection of the first wave dominant follicle in heifers, and that both are reliable markers to predict which follicle in a growing cohort of 5- to 8.5-mm follicles becomes dominant.
Activins↗
Maternal exposure to octylphenol suppresses ovine fetal follicle-stimulating hormone secretion, testis size, and sertoli cell number.
We have tested the hypothesis that maternal exposure to octylphenol, a putative endocrine disrupting chemical, will suppress gonadotropin secretion with a concomitant decrease in testis size and Sertoli cell number during fetal life in the lamb. In Exp 1, pregnant ewes received a continuous iv infusion of diethylstilbestrol (DES; 50 microg/kg x day), octylphenol (1000 microg/kg x day), or vehicle (1:4, alcohol-saline) from days 110-115 of gestation. The fetuses were chronically catheterized in utero, and blood samples were collected every 8 h to monitor gonadotropin secretion. In Exp 2, pregnant ewes received twice weekly sc injections of DES (0.5 microg/kg x day), octylphenol (1000 microg/kg x day), or corn oil from day 70 of gestation to birth. The pituitary gland and testes were collected from the lambs at the end of the treatment period. In Exp 1, maternal exposure to octylphenol suppressed (P < 0.05) FSH concentrations without any effect (P > 0.05) on LH concentrations compared with those in control fetuses. In Exp 2, long-term maternal exposure to octylphenol or a 1000-fold lower dose of DES suppressed (P < 0.05) FSH, messenger RNA levels and the number of FSHbeta-immunopositive cells in the pituitary gland and reduced testis weight and the number of Sertoli cells in the testis compared with those in control lambs. We conclude that maternal exposure to octylphenol inhibits the secretion of FSH in the fetus with a concomitant decrease in testis size and Sertoli cell number at birth.
Animals↗
The effect of acute nutritional change on follicle wave turnover, gonadotropin, and steroid concentration in beef heifers.
The effects of acute nutritional change on endocrine and ovarian characteristics were studied in cyclic (intact; n = 20) and long-term ovariectomized (ovx; n = 18) heifers being fed 1.2 x maintenance (1.2M). On d 7 of an 8-d progesterone and estradiol treatment, intact and ovx heifers were randomly allocated to diets providing .4, 1.2, or 2.0M until emergence of the second follicular wave after ovulation in intact heifers. In intact heifers, two of eight fed .4M failed to ovulate. In the other six, growth rate and maximum diameter (1.1+/-.09 mm/d and 10.1+/-.7 mm, respectively) of the first dominant follicle (DF) postovulation were less (P<.05) than in heifers fed either 1.2 (1.6+/-.18 mm/d; 12.9+/-.44 mm) or 2.0M (1.6+/-.08 mm/d; 12.7+/-.7 mm). In intact heifers, LH pulse frequency and amplitude were not affected by diet (P>.10). In ovx heifers, the frequency of LH pulses was unaffected by diet (P>.10), but heifers fed .4M had a greater pulse amplitude (P<.05) and mean concentration of LH (P<.001) than those fed 1.2 or 2.0M. Plasma concentrations of FSH were greater (P<.05) in ovx heifers fed .4M than in those fed 1.2 or 2.0M and increased linearly with time (P<.01). The FSH concentrations in heifers fed 1.2 and 2.0M were similar (P>.10) and decreased linearly with time (P<.001). In intact heifers, concentrations of FSH preceding follicle wave emergence were greater in heifers fed .4M (P<.001), but basal concentrations were not affected (P>.10). Concentrations of progesterone and estradiol were unaffected by diet (P>.10). Significant diet x ovarian status interactions in plasma IGF-I concentrations existed. Plasma concentrations of insulin increased as the level of nutrition increased, whereas concentrations of NEFA decreased. In conclusion, growth rate and maximum diameter of the DF were decreased by acute nutritional restriction, without affecting the concentration of LH. The magnitude of the FSH increase preceding new follicle wave emergence increased following dietary restriction, but concentrations of FSH were unaffected during the other stages of DF growth. The results of this study may have important implications for the feeding strategies adapted for high-yielding dairy cows in the early postpartum period when feed intake is often physiologically restricted.
Animal Nutritional Physiological Phenomena↗
Historical perspective of turnover of dominant follicles during the bovine estrous cycle: key concepts, studies, advancements, and terms.
This review chronicles the key concepts, studies, advancements and terms that have led to our current understanding of turnover of dominant follicles (growth and atresia) during the bovine estrous cycle. The "two-wave" concept of follicular development was first proposed in 1960, but remained controversial for the next 28 yr. The concept of the "dominant" follicle was adapted to cattle in 1987. By 1988, ultrasound analysis of individual follicles had demonstrated that heifers usually had two or three distinct waves of turnover of dominant follicles during an estrous cycle. From 1992 to 1993, it was established that a transient rise in serum concentrations of FSH initiated each follicular wave, and a decreased episodic secretion of LH was associated with loss of dominance and the end of a nonovulatory follicular wave. In the past decade, numerous intrafollicular growth factors, such as inhibins, activins, and insulin-like growth factors and their binding proteins, have been identified in follicular fluid of individual bovine follicles. In addition, in vitro studies demonstrate that these growth factors could have endocrine, autocrine, or paracrine actions that modify gonadotropin-stimulated follicular growth and differentiation. However, the precise role of intrafollicular growth factors in turnover of dominant follicles has not been defined. We concluded that two or three FSH-stimulated waves of follicular growth usually occur during the bovine estrous cycle, and each follicular wave culminates in development of a single nonovulatory or ovulatory dominant follicle.
Animals↗
Effect of exogenous LH pulses on the fate of the first dominant follicle in postpartum beef cows nursing calves.
Prolonged postpartum anoestrus in beef cows is due to failure of early dominant follicles to ovulate. It is hypothesized that this failure to ovulate is due to inadequate LH pulse frequency. The objective of this study was to determine whether administration of hourly LH pulses would cause the first dominant follicle to ovulate. In Expt 1, 16 cows received either saline (n = 8) or porcine LH (pLH; 50 micrograms h-1; n = 8) as hourly pulses for 3-5 days from the second day of dominance of the first dominant follicle (day 0). In Expt 2, 21 cows received either saline (n = 7), or 50 micrograms pLH (n = 7) or 100 micrograms pLH (n = 7) as hourly pulses for 3 days. Appropriate ovarian scanning and assays of blood samples were carried out. In Expt 1, the number of dominant follicles that underwent atresia was not affected by increasing the number of LH pulses, but the duration of dominance (days) of the first and second dominant follicles and maximum size (mm) of the second dominant follicle were increased (P < 0.05). Oestradiol concentrations were higher (P < 0.05) in cows given hourly pLH pulses (3.1 +/- 1.2 pg ml-1) compared with controls (1.2 +/- 0.2 pg ml-1). Four of eight treated cows had an anovulatory LH surge. The number of follicle waves to first ovulation was not different (P < 0.05) between control (4.6 +/- 0.9) and pLH treated cows (3.9 +/- 0.5). In Expt 2, four of seven cows given pulses of 100 micrograms pLH h-1 ovulated the first dominant follicle, and the interval from calving to first ovulation was decreased (P < 0.05). In the remaining three cows, the duration of dominance of the first dominant follicle was increased (P < 0.005), the maximum size of the first dominant follicle was greater (P < 0.05), and the interval (days) from the start of infusion to new wave emergence was greater (P < 0.05) compared with cows that failed to ovulate in either the 50 micrograms pLH h-1 or control treatments. In conclusion, hourly pulses of pLH from day 1 after dominance of the first dominant follicle in postpartum beef cows can either prolong dominance or induce it to ovulate. This finding supports the hypothesis that LH pulse frequency is a key determinant of the fate of the dominant follicle in the early postpartum period.
Analysis of Variance↗
Effect of acute nutritional restriction on incidence of anovulation and periovulatory estradiol and gonadotropin concentrations in beef heifers.
The effects of acute nutritional restriction on follicular dynamics, incidence of anovulation, and periovulatory estradiol and gonadotropin concentrations were studied in two replicates using beef heifers exhibiting regular estrous cycles. Heifers fed a diet supplying 1.2 maintenance (1.2 Mn) were synchronized using an intravaginal progesterone-releasing device for 8 days. One day before device removal, heifers were allocated randomly, within replicate, to a diet supplying 0.4 Mn (n = 20), or kept at 1.2 Mn (n = 21). On the sixth day after detected ovulation, heifers received 500 microg of synthetic prostaglandin F(2alpha) (PGF(2alpha)) to induce luteolysis, estrus, and ovulation of the first dominant follicle (DF). Animals were inseminated and returned to a diet of 1. 2 Mn. Pregnancy diagnosis was performed 30 days later. The maximum diameter subsequently attained by the DF present at progesterone withdrawal was smaller (P < 0.01) in heifers fed 0.4 Mn. Two heifers fed 0.4 Mn failed to ovulate this DF (P > 0.10). Growth rate (P < 0. 01) and maximum diameter (P < 0.001) of the DF in the first follicular wave of the next estrous cycle was also reduced in heifers fed 0.4 Mn. After prostaglandin administration, a further 10 heifers fed 0.4 Mn failed to ovulate the first DF of this cycle, and it regressed (P < 0.001), causing anovulation in 12 of 20 heifers within 13-15 days (P < 0.001). Anovulation of the DF present at progesterone withdrawal was preceded by a proestrous estradiol increase but absence of a gonadotropin surge (2 of 2 heifers), while neither endocrine event was detected before anovulation of the DF of the first new follicular wave (2 of 2 heifers). In cases in which ovulation of the first DF of the new cycle occurred, fertility was similar (P > 0.10) in heifers fed either 0.4 (n = 7) or 1.2 Mn (n = 20). In conclusion, acute nutritional restriction of cyclic heifers from 1.2 to 0.4 Mn decreased the growth rate and maximum diameter of DFs and induced failure of the DF to ovulate in 60% of heifers, but, within the confines of limited animal numbers, did not compromise fertility in heifers that ovulated.
Animal Nutritional Physiological Phenomena↗
Effect of dominant follicle persistence on follicular fluid oestradiol and inhibin and on oocyte maturation in heifers.
The aim of the present study was to characterize in detail the cytoplasmic and nuclear morphology of cattle oocytes recovered from follicles that are dominant for more than 9 days (with low fertility after ovulation), and to relate morphological changes to intrafollicular markers of follicle health. Beef heifers received prostaglandin F2 alpha and a synthetic progestagen (3 mg Norgestomet) for 2 or 10 days on the first day of dominance of the second dominant follicle (DF2) of the oestrous cycle, to give a 4 day (n = 19; N2) or 12 day (n = 21; N10) duration of dominance of the dominant follicle at ovariectomy 18 h after implant removal and before the predicted gonadotrophin surge. Ultrasound scanning determined emergence of a new wave of follicles in five N10 heifers the day before (n = 1) or day of ovariectomy (n = 4) (N10-NonDom). Dominant follicles from the remaining N10 heifers (N10-Dom) were larger (P < 0.05) on the day of ovariectomy (17.8 +/- 0.6 mm) than those from N2 heifers (13.6 +/- 0.4 mm). The oestradiol:progesterone ratio of follicular fluid from N10-Dom heifers was reduced (21.7 +/- 3.1 versus 34.1 +/- 4.4; P < 0.05), while inhibin A (as measured by immunoradiometric assay) was increased (12.7 +/- 1.0 versus 9.0 +/- 0.7 micrograms ml-1; P < 0.05) compared with N2 heifers. Eleven of twelve N2 oocytes demonstrated nuclear activation without germinal vesicle breakdown, while seven of eight N10-Dom oocytes had undergone germinal vesicle breakdown and had progressed to metaphase I (6/8) or II (1/8). In contrast to N2 oocytes, N10-Dom oocytes showed a larger perivitelline space containing more cumulus cell process endings, vacuoles, irregular vesicles, and more mitochrondia and lipid droplets throughout the ooplasm, yet the degree of cumulus cell expansion and atresia was similar. Thus, final oocyte maturation leading to metaphase I is initiated in most dominant follicles with a dominance period of > 9 days before the gonadotrophin surge and is associated with a reduction in dominant follicle health. However, ovulatory ability is maintained and will lead to the ovulation of aged oocytes, markedly reducing subsequent pregnancy rates.
Analysis of Variance↗
Effect of duration of dominance of the ovulatory follicle on onset of estrus and fertility in heifers.
In cattle, prolonged progestogen treatments following luteolysis result in persistent dominant follicles (DF) that are associated with precise onset of estrus but marked reductions in pregnancy rate (PR). The aim was to determine whether increasing duration of dominance of the ovulatory follicle in heifers affected 1) precision of onset of estrus and 2) the timing and nature of the decline in PR. In Exp. 1, duration of dominance of the ovulatory follicle was controlled by causing corpus luteum (CL) regression at emergence of the second follicle wave (mean duration of dominance of 2.1+/-.3 d, Dm2, n = 11) or first day of dominance of the second DF of the cycle; the latter was combined with insertion of a 3-mg norgestomet ear implant for 2 to 10 d to maintain the second DF for 4 (Dm4, n = 32), 6 (Dm6, n = 19), 8 (Dm8, n = 49), 10 (Dm10, n = 28), or 12 d (Dm12, n = 20). Heifers detected in estrus were inseminated approximately 12 h later with frozen-thawed semen. Durations of dominance of the ovulatory follicle of up to 8 d did not affect (P>.05) PR (Dm2 8/9, Dm4 19/28, Dm6 14/18, and Dm8 34/48 heifers pregnant), but PR in Dm10 heifers (12/23 heifers pregnant) was reduced (P = .05) compared with Dm2 heifers; PR in Dm12 heifers (2/17 pregnant) was less compared with all other treatments (P<.01). Fitting a logistic regression model to the pooled PR data to examine the trend in PR showed that extending the duration of dominance from 2 to 9 d and from 10 to 12 d resulted in a predicted decline in PR of 10 to 25% and a further decline of 35 to 75%, respectively. Onset of estrus was delayed in heifers assigned to Dm4 treatment relative to all other treatments (P<.001); it was less variable than that for heifers on Dm6, Dm8, and Dm10 treatments (P<.1). In Exp. 2, heifers received a PGF2alpha analogue and a norgestomet implant on d 12 of the cycle for 3 or 7 d to give approximate durations of dominance of the preovulatory follicle of 2 to 4 d (Dm2-4, n = 29) or 6 to 8 d (Dm6-8, n = 24), respectively. The PR did not differ (P>.05) between heifers on Dm2-4 (22/29) and Dm6-8 (15/24) treatments, but the interval to onset of estrus was delayed (P<.05) by 7 h in the Dm2-4 heifers. In conclusion, restricting the duration of dominance of the preovulatory follicle to < or =4 d at estrus, results in a precise onset of estrus and a high PR following a single AI at a detected estrus.
Animals↗
Regulation of follicle waves to maximize fertility in cattle.
Cattle have recurrent follicular waves every 7-10 days in most physiological situations; an FSH increase is associated with emergence of the wave and LH pulse frequency determines the fate of the dominant follicle. To control oestrus with hormones it is necessary to ensure that either induced corpus luteum regression or the termination of a progestogen treatment coincides with the selection of the dominant follicle during the wave, to give a precise onset of oestrus and high fertility. The exogenous administration of progesterone or progestagen blocks the normal turnover of the dominant follicle once the corpus luteum regresses. Thus, the effects of duration of dominance of the preovulatory follicle on onset of oestrus and fertility were examined. The variation in onset of oestrus was reduced but occurred 5-9 h later after 4 versus 8 days of dominance; pregnancy rate was also affected with dominance periods of 2-4, 4-8 and > 10 days resulting in 0, 10-15% or 20-50% reduction in pregnancy rates, respectively. The necessity for short duration of dominance of the preovulatory follicle means that to ensure high fertility the follicular wave needs to be regulated when using hormones to control oestrus. Two approaches were examined, namely the use of GnRH or oestradiol at time of progesterone intravaginal releasing device insertion. The effect of 250 micrograms of synthetic GnRH on the fate of an existing follicle wave was to ovulate the dominant follicle (20/20 cows) and a new wave emerged 1.6 +/- 0.3 days later; however, there was no effect of GnRH on the wave if administered before dominant follicle selection. The effect of oestradiol concentrations on suppression of FSH in ovariectomized heifers showed that increasing oestradiol to 10-15 pg ml-1 caused a 37 +/- 6.9% decrease in FSH for 24 h, with a subsequent increase to pretreatment values by 57 +/- 13 h. In cyclic heifers, increasing oestradiol to > 10 pg ml-1 in conjunction with progesterone treatment at emergence of the first wave of the cycle affected the current follicle wave by either preventing dominant follicle selection or decreasing diameter of the dominant follicle, without consistently affecting the interval to new wave emergence. Increase of oestradiol after dominance, however, delayed new wave emergence by 2-5 days. A better understanding of the hormonal control of follicle waves will lead to development of improved hormonal regimens to control oestrus sufficiently to give high pregnancy rates to a single AI without recourse to detection of oestrus.
Animals↗
Anaphylactic shock caused by ticks (Ixodes ricinus)
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Adult↗
The effects of dose and duration of administration of pFSH during the first follicular wave on the ovulation rate of beef heifers.
Four experiments were carried out to examine the effects of administration of pFSH (Vetrepharm) from Day 3 of the estrous cycle in conjunction with PG on Day 5 on follicular populations and ovulation rate in heifers. In Experiment 1, 47 heifers were allocated to 1 of 4 treatment groups (n = 11 to 12 per group): a) control, b) 1.5 mg pFSH, c) 2.0 mg pFSH or d) 2.5 mg pFSH until estrus. Heifers assigned to the 3 treatments had a higher ovulation rate than the controls (P < 0.05). In Experiment 2, 45 heifers were allocated to 1 of 5 treatment groups (n = 8 to 10 per group): a) control, b) 1.0 mg pFSH until PG, c) 1.0 mg pFSH until estrus, d) 1.5 mg pFSH until PG or e) 1.5 mg pFSH until estrus. From Day 5, heifers assigned to pFSH treatments had more large follicles than the controls (P < 0.05). There was no effect of treatment on the incidence of twin ovulations. In Experiment 3, 43 heifers were assigned to 1 of 3 groups (n = 11 to 16 per group): a) control, b) 1.0 mg pFSH until estrus or c) 1.5 mg pFSH until estrus. At slaughter, 14 d after administration of PG, the incidence of twin ovulations was 0/11, 7/16 and 8/16 for Groups a, b and c, respectively (P = 0.011). In Experiment 4, pFSH (1.5 mg) was administered to 3 groups during the development of the first dominant follicle: a) growth phase (n = 19); b) static phase (n = 17); and c) decline phase (n = 17). All pFSH-treated heifers had a higher ovulation rate than the controls (P < 0.05); heifers assigned to Group c had a higher ovulation rate than those in Groups a or b (P < 0.05). More heifers assigned to Group c (7/17) superovulated than in the other 2 groups (P < 0.05). In conclusion, administration of 1.0 or 1.5 mg pFSH twice daily beginning at Day 3 of the estrous cycle in association with the induction of luteolysis increased the ovulation rate significantly when pFSH treatment was continued to onset of estrus. The ovulation rate and the occurrence of multiple ovulations were significantly higher when pFSH was administered at the time that the first dominant follicle was in decline.
Animals↗
Resumption of follicular waves in beef cows is not associated with periparturient changes in follicle-stimulating hormone heterogeneity despite major changes in steroid and luteinizing hormone concentrations.
To test the hypothesis that emergence of follicle waves postpartum is associated with a change in circulating FSH isoform distribution, 10 Limousin-cross suckler cows were blood sampled daily from 5 wk prepartum until first ovulation postpartum for FSH, LH, estradiol (E2), and progesterone assay. Follicular growth was monitored daily by ultrasonography from Days 5 to 10 postpartum until first ovulation. Distributions of circulating FSH isoforms were characterized (n = 4 per group) by chromatofocusing at 1) 18-33 days prepartum, 2) 3-5 days prepartum, 3) the first postpartum FSH rise responsible for emergence of the first follicle wave, and 4) the FSH rise that stimulated the ovulatory follicle wave. The interval to detection of the first postpartum dominant follicle (DF) was 9.6 +/- 0.58 days. The number of DF before first ovulation was 2.1 +/- 0.18, and first ovulation occurred at 28.6 +/- 1.54 days postpartum. Serum E2 concentrations were higher (p = 0.0001) in cows during the 5-wk period prepartum (53.8 +/- 6.29 pg/ml) than in the postpartum period up to first ovulation (1.5 +/- 0.15 pg/ml). In late pregnancy, there was an absence of recurrent FSH rises and LH concentrations were decreased (p < 0.0001) compared with those in the postpartum period. The emergence of each follicle wave postpartum was preceded by a 2- to 4-day rise in FSH concentrations. The pattern of FSH isoform distribution did not differ (p > or = 0.75) between the pre- and postpartum periods.
Animals↗
Effect of calf isolation on follicular wave dynamics, gonadotropin and metabolic hormone changes, and interval to first ovulation in beef cows fed either of two energy levels postpartum.
The effects of postpartum energy intake, restricted suckling, and cow-calf isolation on concentrations of LH, FSH, growth hormone, and insulin-like growth factor-I (IGF-I) and on postpartum anestrous interval were determined by randomly allocating beef cows with a mean body condition score of 2.3 +/- 0.1 to receive either 80 MJ metabolizable energy (low-energy diet [L]; n = 51) or 120 MJ metabolizable energy (high-energy diet [H]; n = 52) per cow per day from calving. At 30 days postpartum, cows within diet were randomized to 1) have continued full access to their calves from birth to weaning (ad libitum suckling: ADLIB), 2) be suckled once-daily with their calves penned adjacent (restricted suckling, adjacent: RESADJ), 3) be isolated from all calves except for a once-daily suckling period (restricted suckling, isolated: RESISO). The mean postpartum interval was similar (p > 0.10) for L and H cows (62 and 63 days, respectively). RESADJ cows had a shorter (p < 0.05) postpartum interval than ADLIB cows, and RESISO cows had a shorter interval (p < 0.05) than RESADJ cows, with all effects independent (p > 0.10) of diet. FSH secretion pattern was not affected by diet, suckling treatment, sequential follicle wave number, or follicle wave retrospectively realigned to emergence of first ovulatory wave. Within 5 days of suckling restriction and calf isolation, the number of LH pulses increased from 0.18 to 0.48 pulses per hour (p < 0.05). Both mean LH and the mean number of LH pulses increased linearly (p < 0.01) during the six follicle waves up to the first ovulatory wave. From 80 days before, until the time of, first ovulation, growth hormone decreased (p < 0.05) while IGF-I increased (p < 0.05), irrespective of treatment. The results indicate that the "suckling effect" in beef cows is the major factor affecting the duration of the postpartum interval and suggests that the maternal bond is more important than suckling in regulating LH pulse frequency, the key endocrine factor determining whether or not a dominant follicles ovulates. Removal of the suckling effect resulted in a rapid increase in LH pulse frequency, which was not dependent on level of postpartum nutrition, at least within the nutritional limits of this study. Mean concentrations of FSH, unlike LH, did not vary with follicle wave number, suggesting that lack of FSH is not a major factor delaying the resumption of ovulation in postpartum beef cows.
Animals↗
Testosterone, LH and FSH episodic secretory patterns in GnRH-immunized bulls.
The objective was to determine: (i) the secretory patterns of LH, FSH and testosterone in bulls, and the temporal relationships between the pulses of these hormones; and (ii) the effect of GnRH immunization on these parameters. Friesian bulls (n = 72) were given a primary immunization on day 0 (10-week-old) and a booster immunization on either day 28 (n = 36) or day 56 (n = 36) against either a GnRH-human serum albumin (HSA) conjugate (n = 48) or HSA (n = 24; control). On the basis of GnRH antibody titres at a plasma dilution of 1:160, 1 week after booster immunization, 12 GnRH-immunized and six control bulls from each booster-immunized group were selected and allocated to one of three groups: control bulls and bulls with medium and high antibody titres (0.3, 32 and 51% binding, respectively; pooled SED 4.3%). Blood samples were taken from these animals (n = 36) every 15 min for 8 h on three occasions: (i) 2 weeks after booster immunization when bulls were 4-5 months of age (prepubertal); (ii) at 7 months of age (peripubertal); and (iii) at 11 months of age (post-pubertal). Data were analysed by PULSAR, ANOVA and chi-squared test. The mean antibody titre of the high antibody titre group was greater (P < 0.05) than that of the medium antibody titre group in prepubertal bulls only, but the mean antibody titres of both antibody titre groups were greater (P < 0.05) than that of the control bulls at all times. The frequency and amplitude of LH and FSH pulses in the control bulls were greater (P < 0.05) during prepuberty than after puberty. The frequency, amplitude and duration of LH pulses were greater (P < 0.05) in control bulls than those in medium and high antibody titre bulls at prepuberty. The mean and basal concentrations of FSH and the amplitude and duration of FSH pulses were greater (P < 0.05) in the control bulls than in the high antibody titre bulls at prepuberty. The frequency of testosterone pulses was greater (P < 0.05) in the control bulls than in the medium and high antibody titre bulls at peripuberty. The mean and basal concentrations and pulse amplitude of testosterone were greater (P < 0.05) in high antibody titre bulls than in control bulls after puberty. There was a close temporal relationship between LH and FSH (62.5% of LH pulses were followed by FSH pulses within 75 min) at prepuberty in the control bulls but there was no relationship after puberty. The opposite trend occurred in the high antibody titre bulls, that is, there was no relationship between LH and FSH at prepuberty but there was a close temporal relationship after puberty. The temporal relationship between LH and testosterone was closest at peripuberty (86.7%) in the control bulls, but increased in the high antibody titre bulls from 0% at prepuberty to 57.1% after puberty. In summary, there was an age-related decrease in LH and FSH pulse frequency and amplitude and also in the temporal relationships between these hormones in control bulls. Prepubertal GnRH immunization had a suppressing effect on the pulsatile release of LH, FSH and testosterone before but not after puberty. The presence of high amplitude testosterone pulses in the GnRH-immunized bulls after puberty indicates that the long-term tonic release of LH may be sufficient to initiate a late pubertal-type increase in testosterone concentrations.
Analysis of Variance↗
Hypothermic storage of sheep embryos with antifreeze proteins: development in vitro and in vivo.
Antifreeze proteins (AFPs) non-colligatively lower the freezing point of aqueous solutions, block membrane ion channels and thereby confer a degree of protection during cooling. Ovine embryos following prolonged hypothermic storage were used to determine 1) the type and concentration of a group of AFPs that can confer hypothermic tolerance, 2) the storage temperature, 3) the cooling rate, and 4) the in vitro and in vivo viability. In Experiment 1, Grade 1 and 2 embryos produced following superovulation were either cultured fresh (control) or stored at 4 degrees C for 4 d in media containing protein from 1 of 3 sources: Winter Flounder (WF; AFP Type 1); Ocean Pout (OP; AFP Type 3) at a concentration of 1 or 10 mg/ml; or bovine serum albumen (BSA) at 4 mg/ml in phosphate buffered saline (PBS). Following 72 h of culture, the viability rates were not different between controls (18 21 ); BSA (9 15 ); WF at 1 mg/ml (14 15 ); WF at 10 mg/ml (13 15 ) or OP at I mg/n-d (15 21 ), but were decreased (P < 0.05) in embryos stored in OP at 1 0 mg/ml (I 1 20 ). Pooled data showed higher (P < 0.05) viability rates for WF (27 30 ) than for OP (26 41 ) or BSA (9 15 ). There was no effect of protein source on hatching rates, but mean hatched diameters of embryos were lower (P < 0.05) following storage in BSA. In Experiment 2, Grade I to 3 embryos were either cultured fresh or stored for 4 d at 0 degrees or 4 degrees C in 4 mg/n-d BSA or 1 mg/ml WF. Embryos stored in WF at 4 degrees C (WF/4 degrees C) had comparable hatching rates (8 12 ) to that of controls (10 10 ), but embryos in the other treatments (WF 0 degrees C, 5 11 , BSA 4 degrees C, 6 11 and BSA 0 degrees C, 3 10 ) had significantly lower hatching rates (P < 0.01) compared with controls. Hatched diameters were comparable between controls and embryos stored in WF 4 degrees C, but embryos stored in WF 0 degrees C and BSA at both temperatures had smaller diameters (P < 0.05). In Experiment 3, Grade 1 to 3 embryos were either transferred fresh or were stored for 4 d at 4 degrees C in 4 mg/ml BSA or 1 mg/ml WF at different cooling rates (T1, BSA > 2 degrees C/min; T2, WF > 2 degrees C/min and T3, WF < 1 degrees C/min) prior to transfer. There were no differences in the number of ewes pregnant (T1, 10 1 1; T2, 6 10 and T3, 8 10 ) or in the number of viable fetuses recovered per treatment (T1, 14 25 ; T2, 10 1 4 and T3, 15 2 1) to indicate a negative effect of cooling rate or protein on embryo survival. In conclusion, ovine embryos can be stored in WF or BSA at 4 degrees C for 4 d, yielding similar pregnancy and embryo survival rates as fresh embryos following transfer to recipient ewes.
Journal Article↗
Assessment of viability and mitochondrial function of equine spermatozoa using double staining and flow cytometry.
An objective double-staining method was developed to evaluate viability and mitochondrial function of stallion spermatozoa using flow cytometry. Sperm viability was assessed by propidium iodide (PI) exclusion, and mitochondrial function was measured by the intensity of rhodamine 123 (R123) fluorescence. Flow cytometry estimates of sperm viability measured by PI were equivalent (P > 0.05) to estimates made using Hoechst 33258 stain and fluorescent microscopy (% dead: 25 +/- 2.4 vs 21.5 +/- 3.5). The use of both PI and R123 was validated by addition of various proportions of freeze-shocked (membrane damaged) cells to viable spermatozoa. There was a high correlation (r(2) = 0.996) between increased PI positivestained (dead) cells and the number of membrane-damaged spermatozoa added (% dead: 29 +/- 0.4, 44 +/- 1.4, 58 +/- 0.9, 75 +/- 0.7 and 91+/- 0.25 vs 0, 25, 50, 75 and 100% damaged cells, respectively). Optimal mitochondrial activity (OMA), as assessed by R123 uptake, was also reduced proportionally (r(2) = 0.976) by the percentage of membrane-damaged cells added (% OMA: 48 +/- 0.6, 37 +/- 1.7, 29 +/- 0.5, 16 +/- 1, 3.8 +/- 1.3 vs 0, 25, 50, 75 and 100% damaged cells, respectively). The mitochondrial inhibitors rotenone and monensin significantly depressed optimal mitochondrial activity (P < 0.001), and there was a significant positive correlation (r(2) = 0.959) between the dose of inhibitors added and the population of sperm cells exhibiting minimal R123 staining (4 -/+ 0.9, 12 -/+ 1.6, 14 -/+ 0.1 and 28 -/+ 2% for treatments with 0, 0.5, 1 and 2 x 10(-5) M rotenone and 0, 0.5, 1, and 2 x 10(-4) M monensin, respectively). Finally, it was shown that treatments containing identical proportions of membrane-damaged cells yielded similar results in terms of viability and mitochondrial activity, irrespective of whether the staining procedure was single or double (P > 0.05). The results of the double-staining method revealed that the percentage of spermatozoa with optimally functioning mitochondria was significantly correlated with the percentage of viable (PI negative) sperm cells (r(2) = 0.998). Flow cytometric analyses using this staining procedure provides reliable and rapid (10,000 cells/min) qualitative assessment of stallion semen.
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The effect of immunization of heifers against alpha 1-26 inhibin conjugate on the superovulatory response to pFSH.
A total of 121 heifers was blocked by time and diet and then randomly assigned, within block, to an inhibin-immunized (I) or a control (C) group. Immunized heifers (n = 61) received a primary immunization (Day 0) with 0.33 mg of an alpha 1-26 bovine inhibin fragment-human serum albumin (HSA) conjugate injected with non-ulcerative Freund's and DEAE-dextran adjuvants. Booster injections were given on Days 28 and 56. Control heifers (n = 60) received HSA and adjuvants. On Days 56 and 83 the ovaries of heifers were examined by ultrasound to determine the ovulation rate, and blood samples were collected for antibody titer determination. On Day 84, 61 heifers (C, n = 30; I, n = 31) received a total of 24 mg of porcine follicle stimulating hormone (pFSH), while 60 heifers (C, n = 30; I, n = 30) received 12 mg im pFSH, which was administered twice daily for 4 d in decreasing doses during the mid-luteal phase of the estrous cycle. Luteolysis was induced with prostaglandin F(2alpha) analog. The heifers were artificially inseminated and were slaughtered 7 d after estrus. Embryos were recovered and morphologically graded on a scale of 1 to 5 (1 = excellent; 5 = degenerated). Antibody titers (percentage binding at 1:125 serum dilution) differed (P < 0.01) between Group C and I heifers at Days 56 (0.1 vs 30%) and 83 (0.2 vs 37%), and 26% of Group I and 1% of Group C heifers (P < 0.01) had twin ovulations on Day 83. The mean number of embryos recovered was reduced (P = 0.02) in Group I heifers (8.9 +/- 1.2) compared with C heifers (12.1 +/- 1.1); however, the mean number of freezable embryos (Grades 1 and 2) was not affected (P = 0.61) by immunization, and there was no interaction with pFSH (P = 0.36). Ovulation rate as well as embryo yield and quality were not different (P > 0.10) between Group C and I heifers when 12 mg pFSH were administered; however, immunization decreased the superovulatory response to 24 mg of pFSH.
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