M. B. Cunningham
W. F. Pope
The Ohio State University
Department of Animal Sciences
Seventy-eight gilts were utilized in two experiments to examine if one mg. of testosterone, given coincident with follicular growth during the period of gonadotrophin treatment, could alter the number of accessory corpus luteum (CL) that formed. In the first experiment, pregnant mare serum gonadotropin (PMSG) was given on day 11 and human choronic gonadotropin (hCG) on day 14. In the second experiment, PMSG was given on day four and hCG on day seven. Testosterone or vehicle was injected daily beginning on the day of PMSG injection and continued for six days. When PMSG was administered at a 750 IU dosage, treating gilts with testosterone tended (P < 0.09) to increase the number of accessory CL. Likewise, when PMSG was given on day four, treatment with testosterone increased (P < 0.05) the number of accessory CL. These data suggest that follicular development during the luteal phase in gilts might be dependent on the amount of available testosterone substrate.
Testosterone is utilized by developing follicles as a substrate for estradiol synthesis. Estradiol is requisite for the maintenance and health of growing follicles. A healthier follicle can result in improved oocyte quality. It has been previously demonstrated (Cardenas and Pope, 1994) that treating mature gilts with exogenous testosterone six days before estrus, increased the number of follicles that ovulated and improved the percentage of resulting blastocysts that survived to day 11 (day 0 = the first day of estrus).
No information is available regarding how much follicular development can be affected with exogenous testosterone at earlier stages of the estrous cycle. For example, the question remains: Can follicles on day four, when the follicular wave first begins, utilize testosterone as supplemental substrate? Similarly, since ovulations can be induced during the mid-cycle with gonadotrophins, it remains to be investigated if testosterone can augment follicular development at this time.
A homogeneous group of crossbred Landrace (1/4) x Yorkshire (1/4) x Duroc (1/2) gilts, weighing from 140 to 160 kg, were used in the two experiments. Estrus was detected twice daily (8,300 and 2,000) in the presence of intact boars. Only gilts that previously exhibited estrous cycles of 19 to 21.5 days were included in the experiments.
Gilts were assigned to a two by two factorial arrangement of treatments (10 per group) in order to partition the effects of dose of PMSG from the presence or absence of exogenous testosterone. Gilts were administered vehicle (corn oil, one ml) daily or one mg of testosterone (n = 20) on days 11 to 16 of the estrous cycle. Accessory ovulations were induced by administering, to all the gilts, PMSG and hCG on days 11 and 14, respectively. The hCG dose was always the same ( 750 IU per gilt) but the PMSG dosage was either 750 or 1,500 IU per gilt (n = 20).
As induced corpora lutea will extend the luteal phase (Neill and Day, 1964; Caldwell, et al., 1969; Christenson and Day, 1971), the number of accessory CL were counted following a midventral surgery on day 23. The accessory CL appeared equivalent to normal day seven CL with respect to their size, color, and degree of vascularity. The CL were formed initially in the estrous cycle (spontaneous not induced) were smaller, pale colored, indicative of luteolysis, and were not counted as accessory CL. The experimental protocol allowed for the exclusion of any gilts that came into estrus before day 23 but all the gilts failed to display estrous activity before day 23.
Thirty-eight gilts (18 per group) were treated with vehicle or one mg of testosterone on days four to nine of the estrous cycle. All gilts were induced to ovulate accessory CL following an injection of PMSG (750 IU) and hCG (750 IU) on days four and seven, respectively. In a preliminary trial these superinduction procedures, if started on day three, failed to produce accessory CL. On day 12 of the cycle, the ovaries were surgically examined and the number of accessory CL counted. Accessory ovulations were estimated to have occurred 40 hours after the hCG injection (Dziuk and Baker, 1962; Hunter, 1972) which would have been equivalent to day nine in this experiment. There were two types of CL observed on day 12, the spontaneous CL which were large, contained within their capsule and rigid, versus the accessory CL being considerably smaller, hemorrhagic to red in color and soft. All gilts expressed these two types of CL and none of the gilts came into estrus before day 12.
Data regarding the number of accessory CL were compared by use of a two-way and one-way analysis of variance for a completely randomized design in Experiment 1 and 2, respectively (Steel and Torrie, 1980). Means were compared to one another using orthogonal contrasts. These analysis were performed using SYSTAT (Wilkinson, 1990).
Gilts induced to ovulate during the mid-luteal phase of the estrous cycle were responsive to PMSG (Table 1). The 1,500 IU dosage resulted in more accessory CL than the 750 dosage; however, this response was more variable than the smaller dose. No effect of testosterone could be detected relative to the vehicle treated gilts. When gilts were treated with 750 IU of PMSG the response was less variable and treatment with testosterone tended (P < 0.09) to increase the number of accessory CL.
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Table 1. Mean (+SEM) Number of Accessory CL on Day 23 Following Treatment with Vehicle or Testosterone Before Ovulation in Gilts (n = 100) Induced to Ovulate with PMSG (750 or 1500 IU) and hCG (750 IU) on Days 11 and 14, Respectively. | |||
|---|---|---|---|
| Dose of PMSG | Treatment | No. of CL | Probability |
| 750 | Vehicle | 15.7 (2.4) | |
| Testosterone | 22.4 (2.6) | P < 0.09 | |
| 1,500 | Vehicle | 63.2 (11.7) | |
| Testosterone | 83.2 (12.6) | P < 0.27 | |
Inducing gilts with PMSG on day four resulted in less ovulations than when superinduction was initiated on day 11 (Experiment 1). Exogenous testosterone, during the period of gonadotrophin stimulation, increased (P < 0.05) the number of accessory CL (Table 2). Perhaps, the larger sample size in Experiment 2 accounted statistically for the more pronounced effects of testosterone between Experiments 1 and 2.
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Table 2. Mean (± SEM) Number of Accessory CL on Day 12 Following Treatment with Vehicle or Testosterone Before Ovulation in Gilts (n = 20) Induced to Ovulated with PMSG (750 IU) and hCG (750 IU) on Days 4 and 7, Respectively. | |||
|---|---|---|---|
| Dose of PMSG | Treatment | No. of CL | Probability |
| 750 | Vehicle | 5.9 (1.1) | |
| Testosterone | 11.9 (2.5) | P < 0.05 | |
It has been previously demonstrated that daily exposure to exogenous testosterone, beginning on day 13 (Cardenas and Pope, 1997) or on day 17 (Cardenas and Pope, 1994 ) and continuing until estrus on day 18, respectively, increased the number of follicles that ovulate spontaneously at estrus. This experiment demonstrated that follicular development and subsequent induction of ovulation during the luteal phase was also improved with exogenous testosterone.
Certainly this was not the first experiment to demonstrate that the number of accessory ovulations is dependent on the dosage of exogen-ous gonadotrophin. In the first experiment, the 1,500 IU dose of PMSG resulted in more ovulations than the 750 dosage, but the variability among gilts was substantial, ranging from 29 to 138. Either exogenous testosterone did not affect follicular development of such a large number of follicles or the variability in the number of CL precluded the opportunity to observe an effect of testosterone treatment.
The observation that the number of accessory CL was smaller when PMSG was injected on day four than day 11, although not specifically addressed in this experiment, may be significant when examining the effects of exogenous testosterone. In a preliminary trial, accessory CL could not be induced on day three indicating that follicular development does not begin until day four. This lack of response on day three is contrary to the observations that accessory CL were produced when PMSG when given on day one and two (Caldwell et al., 1969; and Christenson and Day, 1971, respectively), and hCG 72 hours later. Regardless, normal ovulation occurs on day two, and the small post-ovulatory rise in follicle stimulating hormone (FSH) on days three and four (Van de Wiel et al., 1981) could account for the initiation of a wave of follicular development for the subsequent estrus (Dailey et al., 1976; Van de Wiel et al., 1981; Xie, 1989). The observation that the effects of testos-terone were most pronounced in the second experiment relative to the first experiment might be explained because these hormone therapies were coincident with the first, and rather coordinated, initiation of post-estrous follicular growth (Van de Wiel et al., 1981).
Previous experiments have suggested that exogenous testosterone increased the concentration of follicular estradiol (Cardenas and Pope, 1994). An increase in estradiol was speculated to improve follicular health and to improve the health of the oocyte and resulting embryo. The hypothesis that exogenous testosterone was utilized as a source of more substrate for follicular estradiol synthesis was not examined in the present experiment and can only be speculated to be the mechanism for how testosterone increased the number of accessory CL. It was previously suggested that during proestrus the amount of follicular synthesis of estradiol could be a limiting factor to follicular growth. The significance of this investigation and the previous observations might be to suggest that estradiol synthesis might be limiting in follicular development in gilts throughout the estrous cycle.
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