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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
Department of Animal Sciences, The Ohio State University, Columbus 43210
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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Key Words: early weaning • follicle • heifer • ovary • puberty
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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; Byerly et al., 1987; Bagley, 1993). In addition, the probability of heifers reaching puberty before the breeding season has been shown to increase as BW at weaning or growth rate from birth to weaning, or both, increases (Wiltbank et al., 1966; Arije and Wiltbank, 1971; Buskirk et al., 1995).
Wave-like patterns of follicular development have been observed in heifer calves as early as 2 wk of age (Evans et al., 1994b). The maximum diameter of dominant follicles continues to increase through the peripubertal period up to puberty (Bergfeld et al., 1994; Evans et al., 1994b). Serum estradiol concentration has been documented to increase as first ovulation approaches in beef heifers (Evans et al., 1994a), and Melvin et al. (1999) demonstrated that the concentration of estradiol during ovarian follicular waves increases from wave to wave as heifers approach puberty.
Wehrman et al. (1996) observed puberty before 300 d of age (precocious puberty) in up to 25% of heifers. We have induced precocious puberty in nearly 90% of heifers with a combination of early weaning and feeding a high-concentrate diet (Gasser et al., 2006, companion paper). In these heifers the peripubertal increase in LH pulse frequency was advanced, corresponding with precocious attainment of puberty. We hypothesized that ovarian follicular development and estradiol production would also be accelerated in heifers that are weaned early and fed a high-concentrate diet to induce precocious puberty. The objective of this experiment was to describe changes in follicular development that lead to the precocious attainment of puberty in heifers. A secondary objective was to test the impact of age at weaning on pubertal age in heifers.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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Twenty-eight March-born, crossbred, Angus and Simmental heifer calves were blocked by age and BW and assigned randomly to be weaned early at 104 ± 2 d of age and 156 ± 3 kg of BW (early weaned; n = 18) or to remain on their dams until a typical weaning age of 208 ± 3 d of age and 276 ± 7 kg of BW (normal-weaned, NW; n = 10). All early weaned heifers were fed a receiving diet for 3 wk after weaning and were assigned to receive a high-concentrate (EWH; n = 9) or a control (EWC; n = 9) diet beginning at a mean age of 125 d. The NW heifers were fed the control diet after weaning. The EWC treatment was designed with the goal of achieving a similar growth rate to that of the NW treatment.
At weaning, heifers were transported to a facility approximately 145 km from the cowherd and were fed in adjacent pens. It was beyond the scope of the experiment to perform ultrasonography on the heifers in the NW treatment before weaning. All diets consisted of whole shelled corn, alfalfa pellets, pelleted soybean hulls, and a supplement that contained ground corn, soybean meal, urea, vitamins, minerals, monensin (Rumensin, Elanco, Indianapolis, IN), and fat (Table 1). Additionally, a daily ration of 1 kg of grass hay per heifer was added to the experimental diets to aid in the prevention of bloat.
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Ovarian Follicle Examination
Daily transrectal ultrasonographic examinations were conducted for periods of sufficient duration to identify one complete follicular wave in each heifer, by using a 7.5-MHz linear array transducer (500 V, Aloka, Wallingford, CT) with an extension handle molded to fit the tranducer and allow for external manipulation. Examinations occurred at mean ages of 126, 161, 196, 224, and 252 d for EWH and EWC heifers, and 224 and 252 d for NW heifers. The number and diameter of all ovarian follicles 
3 mm in diameter were recorded at each observation session by drawing sketches manually. The dominant follicle of each follicular wave was identified as the largest follicle of that wave that was present in the ovaries until emergence of the subsequent follicular wave was detected. The maximum diameter (largest cross-section) of dominant follicles was defined as the largest size attained by the dominant follicles during the wave of follicular development. The mean number of follicles detected per day throughout the follicular wave was also calculated. Emergence of a new wave of follicular development was confirmed retrospectively and defined as the day on which the largest of a growing cohort of follicles was 4 to 5 mm in diameter. Duration of the follicular wave was defined as the time from the first observed emergence to the subsequent emergence of the new wave. Ultrasonographic examinations were discontinued on individual heifers upon attainment of puberty.
Blood Sample Collection
Blood samples were collected daily during periods of ultrasonography to be analyzed for estradiol concentrations and collected weekly beginning at a mean age of 153 (EWH and EWC) or 216 (NW) d until the end of the experiment to be analyzed for progesterone concentration to determine age at puberty and subsequent luteal activity. All blood samples were collected via jugular venipuncture into evacuated tubes containing anticoagulant (EDTA; Vacutainer, Becton-Dickinson, Franklin Lakes, NJ) and centrifuged at 2,785 x g for 20 min immediately after collection, and plasma was harvested and frozen at –20°C until hormone analysis.
Peak estradiol concentrations were defined as the greatest concentration of estradiol detected from daily samples during the ovarian follicular wave. Age at puberty was defined as 7 d before the collection date of the first blood sample that contained >2 ng of progesterone/mL of plasma or 7 d before the collection date of the first of 2 consecutive blood samples with >1 ng of progesterone/mL of plasma (Day et al., 1984). Heifers that reached puberty before 300 d of age were considered to have exhibited precocious puberty.
Hormone Quantification
Concentrations of estradiol were determined using a double-extraction, single-antibody RIA, as reported by Kojima et al. (1992) and previously validated in our laboratory (Anderson et al., 1996). Blood samples collected on the day of peak estradiol concentrations from each wave of follicular development were identified and were all included in a single assay. The average intraassay CV among duplicate samples was 3.8%, and the sensitivity of the assay was 0.7 pg/mL.
Concentrations of progesterone were determined using a commercially available RIA kit (Coat-a-Count, Diagnostic Products Corporation, Los Angeles, CA) as described previously for our laboratory (Burke et al., 2003). The interassay CV were 13.6, 11.5, and 10.8% for standard concentrations of 1.4, 2.5, and 11.9 ng/mL, respectively. The average intraassay CV was 3.7%, and the assay sensitivity was 0.1 ng/mL.
Statistical Analyses
The effects of treatment, mean day of age, and the interaction of treatment and mean day of age on BW, maximum diameter of the dominant follicle, mean number of follicles, duration of the follicular wave, and peak estradiol concentration during the follicular wave were analyzed by ANOVA using the MIXED procedure of SAS (SAS Inst. Inc., Cary, NC), with repeated measures analysis included in the model. The repeated measures model was
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where Yijk = observation of the jth heifer in the ith treatment on the kth day, µ = the overall mean, Ti = the ith treatment, hj:i = the random effect of the jth heifer within the ith treatment [hj:i ~ N(0,
2h)], Dk = the kth day, (TD)ik = the treatment x day interaction, and eijk = the random residual effect [eijk ~ N(0, 
)], where is the variance-covariance structure of the residual errors with a first-order autoregressive structure (BW and follicular diameter), or compound symmetry (follicular number, follicular wave duration, and peak estradiol concentration) for repeated measurements within heifers.
The effect of treatment on age at puberty and BW at puberty was analyzed by ANOVA using the MIXED procedure of SAS (Yij = µ + Ti + eij, with notations as defined previously).
To further characterize aspects of precocious puberty, heifers were grouped regardless of treatment into those that experienced precocious puberty (PREC) and those that did not (NON). A preliminary comparison of the heifers from the EWH treatment with those from the EWC and NW treatments that experienced precocious puberty did not detect differences between these groups in any aspect of follicular development. Subsequently, similar models to those described above were used to analyze the effect of group (PREC vs. NON), substituted for treatment in the models.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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. As expected, the mean age at puberty in the EWH treatment (252 ± 9 d) was less (P < 0.05) than for heifers in the EWC and NW treatments (308 ± 26 and 330 ± 25 d, respectively), which did not differ. However, no difference was determined between treatments for BW at puberty (overall mean = 382 ± 13 kg). After detection of the pubertal luteal phase, all heifers continued to exhibit subsequent luteal phases of normal duration as detected from continuous weekly blood sampling.
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). Maximum dominant follicular diameter in the EWH treatment was greater (P < 0.05) than in the EWC treatment at mean age of 196 d (12.9 ± 0.5 and 10.8 ± 0.4 mm, respectively) and greater in the EWH treatment (P < 0.05) than the EWC and NW treatments at mean age of 224 d (13.9 ± 0.4, 12.4 ± 0.4, and 11.1 ± 0.3 mm, respectively). Also at mean age of 224 d, maximum follicular diameter was reduced (P < 0.05) in the NW treatment as compared with the EWC treatment. However, there was no difference detected between the EWC and NW treatments by mean age of 252 d. Across all treatments, mean number of follicles during follicular waves decreased with age (P < 0.05), duration of follicular waves increased with age (P < 0.05), and peak concentrations of estradiol during follicular waves increased to mean age of 224 d (data not shown).
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; Gasser et al., 2006, companion paper). To maintain the objective of describing changes in follicular characteristics associated with precocious puberty, heifers that achieved PREC, regardless of treatment, were grouped together for comparison with NON heifers. In addition to the obvious outcome of a younger (P < 0.01) mean age at puberty (PREC, 254 ± 7 d; NON, 390 ± 12 d) with this grouping, heifers in the PREC group also reached puberty at lighter BW (P < 0.01) than heifers in the NON group (349 ± 13 and 451 ± 14 kg, respectively).
A clear influence of group on the maximum diameter of dominant follicles was evidenced by larger follicles (P < 0.01) throughout the experimental period in the PREC as compared with the NON group (Figure 4). No interaction between group and age on follicular diameter was detected. The mean number of follicles 3 mm in diameter was reduced (P < 0.01) in the PREC group at mean age of 161 d as compared with the NON group (7.1 ± 0.3 and 9.3 ± 0.4, respectively; treatment x group, P < 0.05; Figure 5).
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). Peak estradiol concentrations during follicular waves are shown in Figure 7. Heifers from the PREC group had greater (P < 0.05) peak concentrations of estradiol than heifers from the NON group (2.3 ± 0.5 and 1.4 ± 0.5 pg/mL, respectively). Estradiol concentrations were influenced by age (P < 0.05) and increased to mean age of 224 d across groups of heifers.
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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, companion paper). Unexpectedly, approximately one-half of the heifers that were early weaned and fed a control diet or were weaned at 7 mo of age and fed the same control diet experienced precocious puberty. Whereas the occurrence of spontaneous precocious puberty has been documented (Wehrman et al., 1996), we had previously not observed this great of an incidence in heifers from this herd. This diverse response in terms of age at puberty in the EWC and NW animals likely contributed to the failure to detect a difference in BW at puberty between these females and those in the EWH treatment as had been observed in previous work (Gasser et al., 2006, companion paper). However, even with the elevated incidence of precocious puberty in the control treatments, a greater maximum diameter of dominant follicles was detected in heifers fed the high-concentrate diet after early weaning.
The pattern of ovarian follicle development from birth to spontaneous puberty in heifers has been the focus of multiple studies (for review, see Rawlings et al., 2003). Antral ovarian follicles were not observed at birth in heifers (Desjardins and Hafs, 1969) but were present by 2 to 4 wk of age (Erickson, 1966). Thereafter, the number of antral follicles increased rapidly to peak at 3 to 4 mo of age, declined at 7 to 8 mo of age, and remained static through puberty (Evans et al., 1994a). Increased numbers of antral follicles were accompanied by increased systemic (Evans et al., 1994a) and follicular (Dodson et al., 1989) estradiol concentrations. Follicles grew in waves throughout the period from approximately 2 wk of age to puberty. Diameter of the largest follicle of a corresponding wave increased from 1 to 8 mo of age (Hopper et al., 1993; Evans et al., 1994b) and during the peripubertal period (Day et al., 1987; Bergfeld et al., 1994). The pattern of follicular development from birth to puberty in heifers is reflected in changes in LH secretion during the same period. Pulsatile secretion of LH is established by 1 to 2 mo of age in heifers (Anderson et al., 1986). Thereafter, secretion of LH increases until 3 to 5 mo of age, and then declines, remaining at a relatively low, static level until initiation of the peripubertal increase in LH (Schams et al., 1981; Day et al., 1987; Evans et al., 1992).
Comparisons of heifers that had precocious puberty with those that did not, regardless of treatment, provided a much clearer picture of the changes in follicular development that were associated with attainment of precocious puberty. Diameter of the dominant follicle was approximately 1.5 mm greater, and the duration of the follicular wave was approximately 1 d longer at the first observation of follicular development at mean age of 126 d. Maximal concentrations of estradiol during the follicular wave did not appear to differ at this age, but 35 d later at the second observation, maximum estradiol concentrations during the wave were nominally increased. These differences were largely maintained throughout the experimental period. These observations of differences in follicular development correspond well with the previously detected increase in frequency of LH pulses associated with the attainment of precocious puberty (Gasser et al., 2006, companion paper), suggesting that the peripubertal transition in ovarian follicular development and LH secretion has been shifted to a younger age in instances of precocious puberty
The signals that promote the early activation of the reproductive axis and hence lead to precocious puberty are unknown at present. Potentially, responses to separation from the dam and her milk, increased BW gain, qualitative factors of the high energy diet, or some interaction of these factors could serve as a signal to initiate the process leading to precocious puberty. The present experiment provides valuable insight toward identifying some of these potential factors.
In earlier observations of the effect of early weaning and feeding a high-concentrate diet on precocious puberty in heifers (Day and Anderson, 1998; Gasser et al., 2006, companion paper), it was not possible to separate the relative contributions of weaning age and diet to the induction of precocious puberty. Thus, in the current study, the control diet was formulated to result in BW gains in early weaned heifers that were approximately similar to the expected BW gains for contemporary heifers that remained with their dams until weaning at 7 mo of age. Body weights were nearly identical for the EWC and NW treatments throughout the experiment. Comparison of these treatments permitted the evaluation of the impact of separation of the calf from its dam on age at puberty and characteristics of follicular development without differences in BW.
Mean age and BW at puberty did not differ between EWC and NW heifers, and a similar proportion of heifers in both treatments achieved precocious puberty, suggesting the progression of the process of sexual maturation was similar within these treatments. Thus, in heifers growing at a typical rate, large differences in timing of weaning and compositional differences in the diet (EWC, control diet; NW, milk and pasture) did not appear to influence the pubertal process in these 2 treatments. A difference in maximum diameter of dominant follicles was detected between the NW and EWC treatments at mean age of 224 d. The significance of this difference is difficult to interpret because this observation was taken less than 3 wk after weaning. Considering the stress associated with weaning, it is difficult to determine whether the observed difference was real or simply due to the stress of weaning and adjustment to a new diet, especially because the subsequent observation 28 d later revealed no differences between these treatments.
A treatment consisting of heifers with a similar rate of BW gain to the EWH treatment but weaned at a normal age was not included in this experiment. Therefore, the relative contributions of early weaning and diet composition at the greater rate of BW gain and energy content of the diet cannot be determined. Interestingly, the EWH treatment appeared to induce an increase in follicular diameter very rapidly, as seen at the first examination period after initiation of dietary treatments. This rapid effect occurred before the divergence of BW in heifers, signifying that it may have been accomplished through a more direct effect of the diet. Though the mechanism by which this occurred is unknown, one possibility is that increased dietary energy intake enhanced the responsiveness of the ovaries to gonadotropin stimulation through components of the somatotropic axis. Gong (2002) has reviewed evidence that hormones such as GH, insulin, and IGF-I are not only important regulators of metabolic processes, but they also play an important role in follicular development in cattle. Bergfeld et al. (1994) also observed a rapid increase of follicular diameter within the first month after prepubertal heifers were fed an increased energy diet.
An interesting finding was the greater than expected incidence of precocious puberty that was exhibited by the EWC and NW heifers. Pasture feed resources preceding the time of early weaning were excellent during the year this experiment was conducted, and the EWC and NW heifers from the present experiment were approximately 35 kg heavier at mean age of 103 d than similar control heifers at the same age in our previous work (Gasser et al., 2006, companion paper). We speculate that the nutritional plane provided to these heifer calves and their dams during their first months of life, before initiation of this experiment, was a potential source of stimulation for advancement of early reproductive development. This early influence may have potentiated the effectiveness of subsequent dietary treatments for advancement of puberty in all heifers. Ferrell (1982) suggested that increased preweaning growth rate in calves may be the reason that cattle from breeds selected for increased milk production also tend to reach puberty at a younger age. Madgwick et al. (2005) reported recently that exogenous treatment with GnRH only from 4 to 8 wk of age hastened puberty in heifers. It appears that environmental influences during early life in heifers can have a pronounced effect on future reproductive development.
In the present experiment, mean number of follicles was reduced at mean age of 161 d in heifers that experienced precocious puberty. The reason for this effect is not clear. However, because the duration of follicular waves was extended in heifers that attained precocious puberty, and follicle dominance results in fewer follicles present, the reduction in mean follicular numbers during the follicular wave could potentially be caused by an extended period of follicle dominance associated with the extended follicular wave. The mechanism by which follicular wave duration is extended at this age is not known. Erickson (1966) observed that the number of follicles in female cattle increased between birth and approximately 6 mo of age, and then decreased thereafter. It is conceivable that the heifers that attained precocious puberty progressed to the point of decreasing follicular numbers at an earlier age.
Peak concentrations of estradiol were elevated in heifers that experienced precocious puberty, and mean concentrations increased to mean age of 224 d. Heifers that did not attain precocious puberty appeared to display more fluctuation in estradiol concentrations, including a numerical decrease at mean age of 252 d (see Figure 7
). Similarly, Evans et al. (1994b) noted a numerical increase and decrease in estradiol concentrations during this same age in heifers. These heifers may be entering into a static phase of development, as proposed by Day and Anderson (1998) to occur between 5 and 10 mo of age in heifers that reach puberty around 12 mo of age, during which time LH secretion remains relatively low due to restraint by estradiol negative feedback. In contrast, at this same age the heifers that experienced precocious puberty in the current study appear to maintain elevated concentrations of estradiol as they progress toward the precocious attainment of puberty.
Results from this experiment, considered together with evidence from Gasser et al. (2006, companion paper), support the hypothesis that processes associated with the peripubertal phase of development leading to puberty are shifted back to an earlier age to facilitate precocious attainment of puberty in heifers that are weaned early and fed a high-concentrate diet. The mechanism by which this shift is accomplished is not yet understood. This method of early weaning and feeding a high-concentrate diet appears to stimulate physiological mechanisms in such a manner as to allow heifers to overcome the estradiol negative feedback on LH secretion (Day and Anderson, 1998) at a younger age. This may be accomplished directly through increased stimulation of gonadotropin secretion or indirectly through hastening the decline in estradiol negative feedback. Further research is needed to determine whether changes in negative feedback of estradiol on secretion of LH are associated with the hastened activation of peripubertal processes, as well as to better understand control of the timing of these processes and the potential effects of this treatment on lifetime reproductive performance.
In conclusion, the precocious attainment of puberty was effectively induced in heifers that were weaned early and fed a high-concentrate diet from weaning through puberty. Precocious puberty also occurred in approximately half of the heifers fed the control diet, regardless of age at weaning, suggesting a potential influence of nutritional status during the first months of life in heifers, before the initiation of treatments in this experiment. Heifers that experienced precocious puberty did so at lighter BW than other heifers. An apparent shift of the peripubertal maturational period to an earlier age was associated with precocious attainment of puberty in heifers. This was evidenced by advanced increases in maximum diameter of dominant follicles, duration of follicular waves, and peak estradiol concentrations displayed during follicular waves.
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IMPLICATIONS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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Footnotes |
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2 Current address: Southern Utah Univ., 351 W. University Blvd, Cedar City, UT 84720.
3 Current address: Dexcel Limited, Hamilton, NZ.
4 Current address: Univ of Nebraska, Lincoln, NE 68528.
5 Corresponding author: day.5{at}osu.edu
Received for publication November 3, 2005. Accepted for publication April 5, 2006.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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  C. L. Gasser, E. J. Behlke, D. E. Grum, and M. L. Day Effect of timing of feeding a high-concentrate diet on growth and attainment of puberty in early-weaned heifers J Anim Sci, November 1, 2006; 84(11): 3118 - 3122. [Abstract] [Full Text] [PDF]
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C. L. Gasser, D. E. Grum, M. L. Mussard, F. L. Fluharty, J. E. Kinder, and M. L. Day Induction of precocious puberty in heifers I: Enhanced secretion of luteinizing hormone J Anim Sci, August 1, 2006; 84(8): 2035 - 2041. [Abstract] [Full Text] [PDF]
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C. L. Gasser, G. A. Bridges, M. L. Mussard, D. E. Grum, J. E. Kinder, and M. L. Day Induction of precocious puberty in heifers III: Hastened reduction of estradiol negative feedback on secretion of luteinizing hormone J Anim Sci, August 1, 2006; 84(8): 2050 - 2056. [Abstract] [Full Text] [PDF]
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