Effect of teasing by the ram on the onset of puberty in Romney ewe lambs

Abstract

In two consecutive years (2008 and 2009), ewe lambs that had not attained puberty were allocated to one of two groups (Unteased [n=50 in both years] or Teased [n = 50 in 2008 and n=49 in 2009]) for 17 days (D1 to D17). In 2009, the ovaries of all ewe lambs were examined on D5, D9 and D12 by transrectal ultrasonography. From D18 to D28, crayon-harnessed teaser rams were introduced to both groups each year. In both years a greater proportion (P < 0.05) of Teased than Unteased ewe lambs achieved puberty (based on progesterone concentrations) during D1 to D17 and displayed oestrus during D18 to D28. Teased ewe lambs had larger (P < 0.05) follicle diameters on D9 and D12 compared with the Unteased but differences in corpus luteum sizes were not found (P > 0.05). We have demonstrated that teasing advances the onset of puberty. In ewe lambs that responded to teasing, it appears the response is somewhat delayed in comparison to that observed in mature ewes.

Keywords:

• puberty
• teasing
• ewe lambs
• hoggets

Introduction

Breeding ewe lambs (hoggets) for the first time at eight months of age is a potential means of increasing ewe lifetime reproductive performance compared with the traditional first breeding age of 18–19 months (two-tooths). However, only a third of ewe lambs are bred in New Zealand each year and their reproductive performance is far lower than that of mature ewes (Anon 2010). Low reproductive performance of ewe lambs is a reason that many New Zealand farmers give for not breeding ewe lambs (Kenyon et al. 2004).

In mature ewes, exposure to vasectomized rams (teasers) just prior to the natural onset of the breeding season is an established method to advance oestrus and therefore the breeding season (Edgar & Bilkey 1963; Knight 1980, 1983; Martin et al. 1986; Rosa & Bryant 2002). The characteristic response of mature ewes to this exposure is an increase in luteinizing hormone (LH) pulse frequency that stimulates follicle growth and oestradiol secretion leading to a LH surge and ovulation within 60 hours (Martin et al. 1986; Rosa & Bryant 2002). The first ovulation is silent and is followed by ewes either displaying a normal luteal phase or a short luteal phase followed by a normal length luteal phase (Martin et al. 1986). This results in a biphasic pattern of oestrus behaviour peaking at approximately days 18 and 24 after exposure to the teaser (Knight 1983; Rosa & Bryant 2002).

It is probable that one reason for the low reproductive performance of ewe lambs is that many have not reached puberty prior to introduction of the entire ram. Therefore, stimulating puberty prior to ram introduction should improve their reproductive performance. It was suggested in 1963 that rams could be used to stimulate puberty in ewe lambs (Edgar & Bilkey 1963). Recently it has been shown that exposing ewe lambs to teasers can increase the percentage of ewe lambs displaying oestrus early in the breeding period, resulting in a more compact breeding and increasing the numbers pregnant (Kenyon et al. 2005, 2006a, 2007, 2008a,b). In these studies, it was not known how many ewe lambs had spontaneously achieved puberty prior to teasing making it difficult to accurately determine the magnitude of the response. Further, there is lack of information on the ovarian response of ewe lambs to the exposure to a teaser. It has been shown that fence-line contact of ewe lambs with a ram resulted in increased follicle size and stimulated the formation of some luteal structures (Bartlewski et al. 2002). Although the ovulation of larger follicles is associated with a larger corpus luteum volume in ewes, that subsequently produce more progesterone (Bartlewski et al. 1999), such a relationship has not been studied in teased ewe lambs.

It was hypothesized that in pre-pubertal ewe lambs that, firstly, teasing would induce puberty and, secondly, that the increase in the proportion of ewe lambs attaining puberty after exposure to teasers would be associated with an increase in the size of the follicle and the volume of the corpus luteum, leading to higher progesterone concentrations. Therefore, the aim of this study was to compare the ovarian response and progesterone profiles of pre-pubertal ewe lambs exposed or not, to teasers 17 days before the beginning of the breeding season.

Materials and methods

Location

The studies were undertaken on the Massey University Tuapaka farm, 15 km southwest of Palmerston North, New Zealand (40°S, 175°E; summer solstice, 21 December; winter solstice, 21 June). The study was conducted with ethical approval from Massey University.

Experimental design and animals

Ewe lamb selection

The same experimental design was repeated in two consecutive years (23 March to 12 May 2008 and 25 March to 10 May 2009). In both years, 160, eight-month-old Romney ewe lambs (weighing 32.3±0.2 kg [SEM] and 35.0±0.1 kg in 2008 and 2009, respectively) were blood sampled via jugular venipuncture (8 ml, lithium heparin, Becton, Vacutainer Systems, US) on days (D) −21, −18, −14, −11, −7 and −4 before the main study. Samples were immediately placed on ice until centrifugation at 905 g for 15 min. Plasma samples were then frozen at −20 °C. Attainment of puberty has previously been described as occurring when progesterone concentrations are greater than 1 ng/ml on two consecutive sampling occasions (Wells et al. 2003; Valasi et al. 2006). To ensure no ewe lambs in the main study had attained puberty, any ewe lambs that displayed progesterone concentrations greater than 1 ng/ml during D-21 to D-4, on just one occasion, were excluded. From the remaining lambs, 100 (2008) and 99 (2009) ewe lambs were selected at D1 and weighed.

Main study—design

At D1, the selected ewe lambs were stratified by weight and randomly allocated to either an Unteased (n=50 in both years and kept away from male sheep for 17 days) or Teased group (n=50 [2008] and n=49 [2009], joined with four [2008] and five [2009] vasectomized Perendale rams [teasers] for 17 days). On D1, D4, D7, D9, D13, D15 and D17 all ewe lambs in both groups were blood sampled. In 2009, the samples were collected on D5 and D12 instead of D4 and D13. On D18, the two groups were merged for an 11-day period (D18 to D28) and crayon-harnessed teaser rams were introduced. The ewe lambs were blood sampled again on D19, D22, D25 and D28. Ewe lambs that displayed crayon marks on their rumps (an indicator of oestrus behaviour) were identified on D19, D25 and D28 in 2008 and D19, D22, D25 and D28 in 2009. This relatively short oestrus measurement period was used because in mature ewes, it is well established that oestrus will generally occur between 17 and 28 days after the first day of teaser exposure (Knight 1983; Martin et al. 1986; Rosa & Bryant 2002). All ewe lambs in the present study were managed under commercial grazing conditions during the experimental period.

Ultrasonography

In 2009, the ovaries of all ewe lambs in both groups were examined on D5, D9 and D12 using a real-time, B-mode scanner (Mindray DS USA Inc, Digiprince DP 6600 Vet, Nanshan, China) with a rigid 7.5 MHz transducer modified for external manipulation in the rectum (Viñoles et al. 2010). For both ovaries on each day, all corpora lutea (CLs) present were counted. The size of the corpus luteum (CL) was measured in its vertical and horizontal dimensions, and the mean diameter used to calculate CL volume, using the formula to calculate a sphere volume (πxd³/6). If a cavity was present, the mean diameter of the two dimensions was calculated, and the volume of the cavity calculated and subtracted to give the total CL volume. The diameter of the largest follicle present was measured on its vertical diameter. The accuracy and precision of this procedure has been confirmed by analyses of scanned ovaries subsequently at post-mortem (Viñoles et al. 2004).

Progesterone concentrations

Puberty in this study was deemed to occur when progesterone concentrations were greater than 1 ng/ml (Wells et al. 2003; Valasi et al. 2006) on two consecutive occasions during the period D1 to D17. Progesterone was measured by RIA using ImmuChem™ Double Antibody Progesterone ¹²⁵ I RIA kit for in vitro diagnostic use, MP Biomedicals, US. The intra-assay coefficients of variation for progesterone were 16.1% (n=10), 8.4% (n=15) and 9.9% (n=10) for low-, medium- and high-quality controls, respectively. The inter-assay coefficients of variation for 14 assays were 16.5%, 14.6% and 18.6% for low-, medium- and high-quality controls, respectively. The assay sensitivity, expressed as ng steroid/ml plasma, was 0.18 ng/ml.

Statistical analysis

Ewe lamb live weight, within year, was analysed using a generalized linear model (Proc GLM, Minitab 13.1, Minitab Inc, State College, PA, US) with the fixed effects of lamb treatment group and whether they obtained puberty (as defined by two consecutive elevated progesterone concentrations above 1 ng/ml) during D1 to D17 and the two-way interaction.

The proportion of ewe lambs in 2009 which displayed a CL at least once on D5, D9 or D12 was analysed as a binomial trait using the SAS (SAS Institute, Cary, NC, US) procedure for categorical data (PROC GENMOD) with ewe lamb treatment as a fixed effect and live weight at D1 was used as a covariate.

The volume of a single CL at D5, D9 and D12 was analysed using a mixed model (PROC MIXED) procedure in SAS with ewe lamb treatment group as a fixed effect and live weight at D1 as a covariate. Only one ewe lamb in the Unteased group had two CLs while 11 in the Teased group had at least two CLs. Therefore, this analysis excluded ewe lambs with more than one CL. Data for binomial traits are present as logit values with back transformed percentages in parentheses.

The diameter of the largest follicle on each ewe lamb in 2009 was analysed with the PROC MIXED procedure in SAS. Ewe lamb treatment, measurement day (D5 vs D9 vs D12) and total CL number identified during the measurement period (0 vs 1 vs 2) were included as fixed effects, with the interaction between measurement day and treatment group tested.

Progesterone concentrations in 2009 at D5, D9 and D12 were analysed with the PROC MIXED procedure in SAS with treatment group and number of CL present (0 vs 1) at each time point as fixed effects. The interactions between these fixed effects were also included in the model. Live weight at D1 was included in the model as a covariate. In total, only one Unteased ewe lamb displayed two CLs during the entire ultrasound measurement period (at D12), whereas 11 Teased ewe lambs displayed at least two CLs (10 had two and one had three). Therefore, due to the unbalanced nature of the data, all ewe lambs with two or more CLs were excluded from the progesterone data analysis at each time point.

The proportion of ewe lambs in 2008 and 2009 which displayed oestrus during D18 to D28 was analysed as a binomial (PROC GENMOD) with ewe lamb treatment as a fixed effect and live weight at D1 as a covariate. The model was then re-analysed including only those ewe lambs that had displayed puberty during D1 to D17.

Results

Progesterone concentrations in the pre-study period

In 2008, only one (35 kg on D-21) ewe lamb from the original 160 had progesterone concentrations greater than 1 ng/ml during D-21 to D1. In 2009, 41 ewe lambs (mean live weight 35.3±0.2 kg on D-21) had progesterone concentrations greater than 1 ng/ml before the beginning of the experiment. These animals were excluded from the main study in 2008 and 2009.

Live weight

Within both years, live weight at D1 did not differ between treatment groups or between ewe lambs that obtained puberty or not during the period D1 to D17 (P > 0.05, Table 1). In addition, there was no interaction (P > 0.05) between treatment group and puberty occurrence for live weight in either year.

Table 1  Live weights of ewe lambs at D1 based on ewe lamb treatment (Unteased vs Teased) and occurrence of puberty (No vs Yes) during the period D1 to D17.

	Live weight (kg)
	2008		2009
	n		n
Treatment
Unteased	50	34.55±0.33	50	36.67±0.20
Teased	50	34.24±0.28	49	36.85±0.26
Puberty^1
No	38	34.32±0.25	24	36.79±0.31
Yes	62	34.49±0.35	75	37.03±0.17
^1 It was deemed that lambs reached puberty when progesterone concentrations were greater than 1 ng/ml on two consecutive occasions during the period D1 to D17 in the 2008 and 2009 studies, respectively.

Proportion of ewe lambs that reached puberty

In both years, a greater proportion (P < 0.05) of Teased ewe lambs achieved puberty in the period D1 to D17 than Unteased ewe lambs (Table 2). These same relationships were still present (P < 0.05) when live weight was used as a covariate in the model (data not shown). In 2009, a greater proportion (P < 0.05) of ewe lambs in the Teased group achieved puberty during the entire period D1 to D28, than in the Unteased group.

Table 2  The effect of ewe lamb treatment (Unteased vs Teased) on the proportion of ewe lambs achieving puberty¹, during D1 to D17 and D1 to D28 periods. Within years and day-intervals, means of treatments followed by different letters are significantly different (P<0.05).

	Proportion that achieved puberty^1
	2008			2009
	n	D1–17	D1–28	n	D1–17	D1–28
Treatment
Unteased	50	−1.15^a ± 0.33 (24.0)^2	0.32 ± 0.29 (58.0)	50	−1.52^a ± 0.37 (18.0)	0.32^a ± 0.29 (58.0)
Teased	50	−0.08^b ± 0.28 (48.0)	0.66 ± 0.30 (66.0)	49	1.36^b ± 0.35 (79.6)	2.73^b ± 0.60 (93.9)
^1 It was deemed that lambs reached puberty when progesterone concentrations were greater than 1 ng/ml (Wells et al. 2003; Valasi et al. 2006) on two consecutive occasions during the period D1 to D28 in the 2008 and 2009 studies, respectively.
^2 Data are presented as logit transformations with back-transformed percentages in parentheses.

The pattern of puberty expression

In 2008, 12 Unteased ewe lambs reached puberty, as indicated by elevated progesterone concentrations during the period D1 to D17. Two of these ewe lambs had elevated concentrations by D7, a further four by D9 and another six at or later than D11. In the Teased group, 24 ewe lambs had elevated progesterone concentrations from D1 to D17. Five of these had elevated progesterone by D7, two by D9 and a further 17 at or after D11.

In 2009, nine Unteased ewe lambs reached puberty during the 17-day period; one ewe by D7, three by D12 and five at D12 or later. In the Teased group, 39 reached puberty from D1 to D17, four by D7, a further six by D9 and another 29 by D12 or later.

Proportion of ewe lambs with corpora lutea identified by ultrasonography in 2009 study

In 2009, a greater (P < 0.05) percentage of Teased ewe lambs had a CL identified at either D5, D9 or D12 (logit value 1.63±0.38 (SEM) (back transformed%, 83.6%) than Unteased ewe lambs (−0.85±0.30, 29.9%). A total of 15 Unteased ewe lambs had a CL identified; of these three were present for the first time at D5, five present for the first time at D9 and seven present for the first time at D12. In the Teased group, a total of 41 had CL and their pattern was 10, 23 and 8 present for the first time at D5, D9 and D12, respectively.

None of the Unteased ewe lambs who had a CL at D5 still had one present at D12. In the Teased group, four of the 10 present at D5 were still present at D12. Three of the five Unteased ewe lambs who had a CL present for the first time at D9 still had them present at D12. In the Teased group, 18 of the 23 ewe lambs who had a CL present for the first time at D9 still had them present at D12.

Diameter of the largest follicle as identified by ultrasonography in 2009 study

There was an interaction (P < 0.05) between ewe lamb treatment and day of observation for follicle diameter, such that, within the Teased ewe lambs, follicle diameter was smaller at D5 (5.2±0.4 mm) than at both D9 (6.9±0.4 mm) and D12 (7.1±0.4 mm), the latter two time points not differing. There was no difference (P > 0.05) across time in the Unteased ewe lambs (5.1±0.5, 5.8±0.4 and 5.4±0.4 mm for D5, D9 and D12, respectively). The diameter of the largest follicle at D9 and D12 differed (P<0.05) between Teased and Unteased ewe lambs.

Volume of CL in single ovulating ewe lambs in 2009 study

Ewe lamb treatment had no effect (P > 0.05) on CL volume on days 5 (Unteased n=2, 306.8±177.8 mm³ vs Teased n=6, 349.8± 177.8 mm³), 9 (Unteased n=4, 170.4±107.0 mm³ vs Teased n=20, 411.0±47.5 mm³) and 12 (Unteased n=5, 145.3±140.3 mm³ vs Teased n=20, 572.0±71.2 mm³).

Table 3 shows that progesterone concentrations were lower (P < 0.05) in Unteased than Teased ewe lambs on D5 and that concentrations were higher (P < 0.05) in ewe lambs that had a CL compared with those that had no CL. Although progesterone concentrations on D5 were below 1 ng/ml in Unteased ewe lambs that had a CL, the concentrations were similar to those from Teased ewe lambs.

Table 3  The effect of ewe lamb treatment (Unteased vs Teased) and corpus luteum number on progesterone concentrations in ewe lambs at D5, D9 and D12 in 2009 study. Within treatments and days means followed by different letters are significantly different (P < 0.05).

	Progesterone concentration ng/ml
	n ^1	D5	n ^1	D9	n ^1	D12
Treatment
Unteased	50	0.37^b±0.19	50	0.62±0.14	49	0.67±0.13
Teased	48	0.67^a±0.12	47	0.81±0.08	39	1.06±0.09
CL Number
0	90	0.28^a±0.06	73	0.33^a±0.06	63	0.45^a±0.07
1	8	0.76^b±0.22	24	1.10^b±0.15	25	1.30^b±0.13
Treat×CL num^2
Unteased×0	48	0.24^a±0.08	46	0.28^a±0.08	44	0.44^a±0.08
Unteased×1	2	0.50^ab±0.38	4	0.95^b±0.27	5	0.91^b±0.24
Teased×0	42	0.31^a±0.08	27	0.37^a±0.10	19	0.47^a±0.12
Teased×1	6	1.02^b±0.22	20	1.24^b±0.12	20	1.66^b±0.12
^1 Table includes only ewe lambs with 0 or 1 CL. Ewe lambs with more CLs are excluded at each time point analysis.
^2 Ewe lamb treatment by CL number interaction.

Proportion of ewe lambs displaying oestrus during D18 to D28

A greater (P < 0.05) overall proportion of Teased ewe lambs displayed oestrus during D18 to D28, than Unteased ewe lambs in both years, regardless of whether they reached puberty or not during D1 to D17 (Table 4).

Table 4  The effect of ewe lamb treatment (Unteased vs Teased) on the proportion of ewe lambs that displayed oestrus during D18 to D28 regardless of whether they reached puberty or not during D1 to D17. Within years, means followed by different letters are significantly different (P < 0.05).

	Proportion displaying oestrus during D18–28
		2008		2009
	n		n
Treatment
Unteased	50	−1.51^1 ± 0.37^a (18.0)	50	−0.95 ± 0.32^a (28.0)
Teased	50	−0.24 ± 0.28^b (44.0)	49	0.63 ± 0.30^b (65.3)
^1 Data are presented as logit transformations with back-transformed percentages in brackets.

In both years, examination of oestrus in only those that displayed puberty during D1 to D17 (as indicated by progesterone concentrations) found no difference (P > 0.05) in the proportion that subsequently displayed oestrus during D18 to D28 (50% vs 75% for Unteased and Teased ewe lambs in 2008 and 89% vs 77% in Unteased and Teased ewe lambs in 2009).

Discussion

The hypothesis that an increase in the proportion of ewe lambs reaching puberty after exposure to teasers is associated with an increase in the diameter of follicles and volume of CL and progesterone concentrations was partially accepted based on the results of the present study. In both years, a greater proportion of the ewe lambs exposed to teasers achieved puberty during the period day 1 to 17 as determined by either progesterone concentration or the presence of at least one corpus luteum. Although teased ewe lambs had larger follicles on two of the three measurement days, they did not have larger corpus luteum volume.

The exposure to the teaser increased the proportion of ewe lambs achieving puberty. Similarly, Al-Mauly et al. (1991) reported that exposure to the ram advanced the onset of puberty as measured by progesterone concentration. The apparent intensity of the puberty onset response during days 1 to 17 was greater in 2009 than 2008. In beef cattle, the pubertal response to the introduction of the bull has been reported to be greater in heavier heifers (Fiol et al. 2010). The 2009 ewe lambs were heavier than their 2008 counterparts, which may suggest a similar relationship to that observed in cattle. Cave (2010) reported, in one of two studies, that ewe lambs of better body condition score were more likely to respond to teasing than those of poor condition. Over the entire 28-day measurement period, there was no difference in the proportion of ewe lambs that had achieved puberty in 2008 but, in 2009, a greater proportion of the teased ewe lambs had achieved puberty. This further indicates the importance of ewe lamb live weight in response to the ram effect. Previous studies have demonstrated that it is often the case that exposure to the teaser induces early breeding activity but may not increase the overall proportion of ewe lambs bred (Kenyon et al. 2006a, 2007, 2008a,b).

The data suggest (based on elevated progesterone concentrations) that the majority of ewe lambs that became pubertal during days 1 to 17, did so at least 9 days after teaser introduction. This pattern differs from that reported for mature ewes in response to ram introduction just prior to the breeding season in that the most common pattern is the occurrence of ovulation within 3 days after ram exposure (Martin et al. 1986; Rosa & Bryant 2002). However, the existence of different patterns of response in mature ewes has been described by Ungerfeld (2007). Delayed ovulations 11 days after the introduction of rams were found in postpartum mature ewes stimulated by the ram effect (Barkawi et al. 1990). It is possible that ewe lambs need a stimulus of longer duration before they respond to the ram effect. In support of this, Kenyon et al. (2008a) reported that ewe lambs exposed to the male for only 2 or 4 days were less likely to be bred in the first 17 days of breeding than those exposed to the male for a full 17 days.

Teased ewe lambs had larger follicle diameters compared with Unteased ewe lambs on days 9 and 12 after ram introduction. The larger follicle diameter is probably explained by a higher LH pulse frequency induced by the males, as has been shown previously (Martin et al. 1986). Interestingly, this was not associated with a larger sized CL which was expected based on previous findings (Gonzalez de Bulnes et al. 2000).

Based on previous studies (Wells et al. 2003; Valasi et al. 2006), puberty was defined as occuring when the progesterone concentration was greater than 1 ng/ml on two consecutive samples. However, Table 3 indicates that progesterone concentrations alone may not be a good indicator of onset of puberty, since pubertal Unteased ewe lambs had a CL that was associated with mean progesterone concentrations less than 1 ng/ml. It has been previously shown that ultrasonography is a highly accurate technique to detect functional CL in adult ewes (Viñoles et al. 2004). These results may suggest that future studies should use a combination of both progesterone concentrations and ultrasonography. A decrease in the cut-off point to define puberty may be an alternative criterion, based on the work by Sunderland et al. (1995) who defined the occurrence of puberty as three consecutive progesterone concentrations above 0.5 ng/ml, with at least one of these above 1 ng/ml.

It is also evident that many ewe lambs, in both the Teased and Unteased groups displayed short luteal phases. Our findings are based on the progesterone profiles and the presence of short-lived CLs in the 2009 study. The occurrence of short luteal phases is a common finding in adult ewes after ram introduction (Rosa & Bryant 2002) and at spontaneous puberty onset and precedes the expression of the first oestrus behaviour (Keisler et al. 1983; Valasi et al. 2006; Ungerfeld 2007).

A greater proportion of ewe lambs exposed to the teaser displayed oestrus during days 18 to 28 in both years. This result supports previous findings (Kenyon et al. 2005, 2006a, 2007, 2008a,b) and explains why a greater proportion of ewe lambs become pregnant early in the breeding period after exposure to the teaser (Kenyon et al. 2006a, 2007, 2008a,b) and display an earlier average lambing date (Kenyon et al. 2006b). Interestingly, examination of only those ewe lambs that displayed puberty, based on progesterone profiles during days 1 to 17, showed that the proportion that subsequently displayed oestrus during days 18 to 28 did not differ between those exposed to the teaser or not. This indicates that the greater percentage of teased ewe lambs that display oestrus early in the breeding period is simply due to the greater number of ewe lambs induced into puberty. It has previously been reported that many ewe lambs can fail to ovulate at their first oestrus (Edey et al. 1997). In the present studies, ultrasound examination was not undertaken during the period of days 18 to 28 although future studies should consider this, as failure to ovulate may explain why many ewe hoggets appear to return to oestrus post breeding (Kenyon et al. 2005, 2006a, 2007, 2008a,b).

By early April, only one ewe lamb in 2008 and 41 in 2009 displayed elevated progesterone concentrations, with a mean live weight of 35 kg. In the following 17 days (D1 to 17), only 12 and nine ewe lambs in the Unteased groups in 2008 and 2009, respectively, achieved puberty. These results suggest it would be difficult to successfully breed ewe lambs weighing less than 35 kg.

Conclusion

It is concluded that teasing ewe lambs advances the onset of puberty. Delayed ovulations are the most common pattern of response after teasing, which may suggest ewe lambs need a longer period of exposure to stimulate appropriate follicle growth. The larger diameter follicles were not associated with larger-sized CL. Teasing increases the proportion of ewe lambs displaying oestrus behaviour 18 to 28 days later.

Acknowledgements

The authors wish to acknowledge Beef + Lamb New Zealand and Massey University for funding this study and the National Research Centre for Growth and Development for part funding PR Kenyon. Advice for the design of the study was given by GB Martin and PAR Hawken and this is acknowledged.