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Corpus luteum size and function following single and double ovulations in non-lactating dairy cows
Theriogenology 67 (2007) 1256–1261 www.theriojournal.com
Corpus luteum size and function following single and double ovulations in non-lactating dairy cows G.E. Mann *, R.S. Robinson, M.G. Hunter University of Nottingham, School of Biosciences, Division of Animal Physiology, Sutton Bonington Campus, Loughborough, LE12 5RD, UK Received 20 September 2006; received in revised form 15 January 2007; accepted 15 January 2007
Abstract Data was collated from a number of studies on various aspects of luteal function in non-lactating dairy cows to allow comparisons to be made between single and double ovulating animals. In these studies, estrous cycles had been synchronized and animals slaughtered on day 5 or 8. The overall incidence of double ovulations was 28.3%. Double ovulation was associated with smaller individual corpora lutea but no difference in total weight of luteal tissue or any aspect of luteal tissue function or plasma concentrations of progesterone. Furthermore, in a sub set of animals, there was no difference in preovulatory follicle characteristics or plasma concentrations of estradiol around ovulation. These results demonstrated a high incidence of double ovulation in nonlactating cows that had no influence on circulating progesterone concentrations. # 2007 Elsevier Inc. All rights reserved. Keywords: Cow; Progesterone; Corpus luteum; Ovulation
1. Introduction Numerous studies have identified the importance of postovulatory progesterone secretion from the developing corpus luteum on the outcome of early pregnancy [1]. While cows are primarily a monovular species and progesterone is normally the product of a single corpus luteum, double ovulations are not uncommon, resulting in two corpora lutea contributing to progesterone secretion. A number of studies in dairy cows have reported twinning rates of between 2.4 and 5.8% [2]. A wide range of factors have been linked to twinning in dairy cows including increasing parity, increased or
* Corresponding author. Tel.: +44 1159 516326; fax: +44 1159 516302. E-mail address: [email protected] (G.E. Mann). 0093-691X/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2007.01.011
decreased milk yield, season, genetics, use of reproductive hormones or antibiotics, ovarian cysts and days open [2–4]. In studies in which ovulation rate has been measured directly, typical reported incidences of double ovulation range from around 5–10% in 1st parity, 10– 15% in 2nd parity and 20–25% in 3rd and subsequent parity cows [4,5]. A recent study [6] reported increased total luteal volume but reduced circulating progesterone 7 days after ovulation in double compared with single ovulating cows. Furthermore, another study [7] also reported lower circulating progesterone concentrations during development of multiple dominant follicles. In view of the importance of progesterone during early pregnancy [8] this association between reduced progesterone and multiple ovulation suggests a potential detrimental effect on pregnancy rate. The aim of this study was to determine the effect of double ovulation compared to single on luteal
G.E. Mann et al. / Theriogenology 67 (2007) 1256–1261
characteristics and plasma progesterone concentrations in non-lactating multiparous Holstein-Friesian cows. 2. Materials and methods In this paper, data from a series of previously published studies [9–11] has been collated and reanalysed to permit comparisons to be made between luteal characteristics and circulating progesterone concentrations in cows exhibiting single or double ovulations. All studies were carried out in multiparous Holstein-Friesian cows obtained from local farms at the end of lactation, and were conducted in accordance with the Animals (Scientific Procedures) Act 1986. The mean age of the animals was 5.5 0.3 years and ranged from 2.4 to 11.0 years. In all cases estrous cycles were synchronized prior to slaughter on day 5 or 8 following estrus.
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cannulated to allow collection of blood samples every 8 h throughout the study for progesterone and estradiol analysis. All samples were collected into heparinized tubes, centrifuged at 1500 g for 10 min and the plasma samples were stored at 20 8C until analysis. 2.1.3. Study 3 In this study the estrous cycles of 12 cows were synchronized by two intramuscular injections of 50 mg of the prostaglandin F2a analogue, cloprostenol (Estrumate) administered 12 days apart. Prior to the second cloprostenol injection, a jugular vein of each cow was cannulated to allow collection of blood samples at 8 h intervals. Samples were centrifuged for 10 min at 1500 g and plasma stored at 20 8C for subsequent hormone analysis. Cows were slaughtered on day 8 of the cycle. 2.2. Assessment of corpora lutea
2.1. Experimental animals 2.1.1. Study 1 In this study the estrous cycles of 17 cows were synchronized by two i.m. injections of 50 mg of the prostaglandin F2a analogue, cloprostenol (Estrumate; Schering-Plough Animal Health Welwyn Garden City, UK) administered 12 days apart. On the morning of day 5 following the day of onset of estrus (day 0), a single blood sample (10 ml) was collected by jugular venepunture, centrifuged (1500 g for 10 min) and plasma collected and stored at 20 8C for subsequent analysis. Cows were then transported to a local slaughter facility where they were killed and the reproductive tract was recovered. Ovaries were examined to confirm the appropriate stage of the cycle and corpora lutea collected and snap frozen in liquid nitrogen. 2.1.2. Study 2 In this study the estrous cycles of 24 cows were initially synchronized by inserting a CIDR device (Pfizer Animal Health, Tadworth, UK) for 10 days and injecting 50 mg of the PGF2a analogue, cloprostenol i.m. (Estrumate) on the day of CIDR withdrawal with a second 50 mg i.m. injection 10–15 days later. In these cows, ovaries were scanned daily by transrectal ultrasonography using a Sonovet 6000 ultrasound scanner with a 7.5 MHz linear array transducer (BCF Technology Ltd., Livingstone, UK) from 3 days prior to the second cloprostenol injection until slaughter on day 5 or 8 of the subsequent cycle. Prior to the second cloprostenol injection, a jugular vein of each cow was
In all studies, the number of corpora lutea was noted and all corpora lutea were dissected free of connective tissue, drained of any fluid and weighed. Tissue was then snap frozen and stored at 80 8C for subsequent determination of progesterone content. In studies 1 and 3, luteal tissue from all animals was finely minced using scalpel blades and washed in phosphate buffered saline by centrifugation at 1500 g for 3 min at 4 8C and the supernatant discarded. Aliquots of approximately 25 mg of tissue ( 2mg) were placed into tubes containing 1 ml of Medium 199 containing 10 mM NaHCO3 1-1, Earle’s salts, 0.068 mM L-glutamine and 25 mM HEPES (Sigma– Aldrich Ltd., Poole, Dorset, UK). Luteal tissue was incubated in control media or media supplemented with 100 ng/ml bovine LH (AFP11743 B biopotency 1.06x oLH NIDDK-I-2). Tissue was incubated for 30 min at 38 8C in a shaking water bath at 70 strokes/min. Following incubation, the supernatant was aspirated and both medium and luteal tissues were snap frozen and stored at 80 8C. In addition, minced tissue was collected prior to incubation to determine initial progesterone content. 2.3. Hormone analysis Plasma progesterone concentrations were measured by radioimmunoassay [12] with a limit of sensitivity of 0.3 ng/ml and the intra- and inter-assay coefficients of variation of 7.3 and 12.6%, respectively. Plasma estradiol concentrations were measured in plasma after diethyl ether extraction using a radioimmunoassay kit
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Table 1 Distribution of single and double ovulating cows across studies and day of slaughter Study
Day of slaughter
Single ovulation (n)
1 2
5 5 8
11 8 12
3
8
Overall
Double ovulation (n) 6 3 1
Incidence (%) 35.3 16.7
7
5
41.7
38
15
28.3
Table 2 Luteal characteristics and plasma progesterone concentration in single and double ovulating cows slaughtered on day 5 and 8 of the estrous cycle Day 5
Day 8
Single (n = 19) Corpus luteum weight (g) Total luteal weight (g) Tissue progesterone (ng/mg) Plasma progesterone (ng/ml) Within days
a,b
P < 0.001; between days
2.9 0.3 2.9 0.3 14.3 1.5 2.0 0.2
a
Double (n = 9) 1.5 0.2 3.0 0.4 13.6 1.4 2.1 0.5
b
Single (n = 19) a,***
6.3 0.5 6.3 0.5 *** 12.8 0.9 6.1 0.5 ***
Double (n = 6) 3.4 0.3b,*** 6.9 0.6 *** 13.7 0.8 5.6 0.7 ***
***
P < 0.001.
(E2 MAIA Serono Diagnostics, Woking, UK) modified for use in the cow [13]. The sensitivity of the assay was 0.5 pg/ml and the intra- and inter-assay coefficients of variation were 13.5 and 13.1%, respectively. Tissue progesterone content was determined following homogenisation and petroleum ether extraction [14] followed by radioimmunoassay as described above. 2.4. Statistical analysis Incidence of double ovulation was analysed by chi-squared test following Yates’s correction. Luteal and follicular characteristics in single and double ovulating animals were compared by analysis of variance with study as a factor. Where repeated progesterone and estradiol concentrations were analysed, repeated sample analysis of variance was undertaken.
summer (28.0%) was not different to that seen during autumn/winter (28.6%). On both day 5 and 8, individual corpora lutea were smaller (P < 0.001) in double ovulating cows though the total weight of luteal tissue was the same as single and double ovulating animals (Table 2). In addition, the progesterone content of luteal tissue was similar in corpora lutea collected from single and double ovulating animals on both day 5 and 8 (Table 2). Thus, the total luteal content of progesterone was also similar between single and double ovulating animal (data not shown). Plasma progesterone concentration on the day of slaughter was also similar between single and double ovulating animals on day 5 and 8 (Table 2). Furthermore,
3. Results The overall incidence of double ovulation across studies was 28.3%. While the incidence was not the same the three studies (Table 1), these differences were not significant. While the incidence of double ovulations was lower in younger (<4.5 years; 3/17–17.6%) than in older (>4.5 years; 12/36–33.3%) cows, this difference was not significant. Furthermore, the mean age of double ovulating cows (5.7 0.6 years) was not different from single ovulating cows (5.4 0.3 years). The incidence of double ovulation during spring/
Fig. 1. Plasma progesterone concentration from day 5 to 8 in single (open circles; n = 19) and double (closed circles; n = 6) ovulating cows slaughtered on day 8.
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Table 3 In vitro progesterone secretion by luteal tissue collected from single and double ovulating cows slaughtered on day 5 and 8 of the estrous cycle Day 5
Basal tissue progesterone synthesis in vitro (ng/mg) LH stimulated progesterone synthesis in vitro (ng/mg)
Day 8
Single (n = 11)
Double (n = 6)
Single (n = 7)
Double (n = 5)
43.4 5.0 50.8 5.8
43.8 5.2 49.0 4.1
40.0 2.6 49.1 3.7
39.0 2.6 47.9 2.5
LH caused an overall stimulation of progesterone secretion (P < 0.01) but there was no effect of ovulation number (n: number of animals from which tissue was incubated).
in cows slaughtered on day 8, plasma concentrations of progesterone from day 5 to 8 were also similar (Fig. 1). In studies 1 and 3, incubation of luteal tissue from single and double ovulating cows slaughtered on day 5 and 8 revealed no difference in basal LH production. Furthermore, while there was a significant stimulation of progesterone secretion by LH, this response did not differ between single and double ovulating animals (Table 3). In study 2, ultrasound scanning allowed assessment of preovulatory follicle development in single and double ovulating animals. There was no significant difference in size of ovulatory follicle at either luteolysis or ovulation (Table 4). Furthermore, there was no difference in the time from luteolysis to ovulation. However, growth rate of the preovulatory follicle tended (P < 0.07) to be higher in single ovulating cows. Measurement of plasma estradiol concentration around ovulation revealed no differences between single and double ovulating animals (Fig. 2). When the weights of individual corpora lutea in double ovulating animals were compared, on day 5 there was no significant difference between the two corpora lutea while on day 8 the smaller corpora lutea were significantly lighter than the larger corpora lutea (Table 5). However, on both days there was a wide spread in the range of differences with some cows having 2 corpora lutea of the same size and others markedly different corpora lutea. This relationship was unaffected by the occurrence of unilateral (n = 10) or bilateral (n = 5) ovulation.
4. Discussion Early studies evaluating the ovulation rate by rectal palpation of number of corpora lutea reported multiple ovulation rates ranging from 5 to 13% [15,16]. More recent studies in high yielding dairy cows have reported rates of 10–39% [4,5,17,18,19] and high milk yield has been suggested as the principle cause of double ovulation in modern dairy cows [3,5]. However, in the present study we have reported a relatively high incidence of double ovulation (28.3%) in non-lactating dairy cows. This suggests that while high milk yield may be involved in the aetiology double ovulation, is not a prerequisite for a relatively high incidence of double ovulation in Holstein-Friesian dairy cows and is unlikely to be a major causal factor. Another causal factor heavily implicated in double ovulation is parity [4,5] and it has been reported that parity determines the incidence of double ovulation independently of milk yield [4]. In the present study, while animal numbers were insufficient to allow robust comparison, the incident of double ovulation in younger cows was lower than in older cows. Other studies, again in lactating animals, have reported associations between double ovulation and reduced circulating concentrations of progesterone [7].
Table 4 Ovulatory characteristics in cows exhibiting single (n = 20) or double (n = 4) ovulation
Follicle diameter at luteolysis (mm) Follicle diameter at ovulation (mm) Growth rate (mm/24 h) Time from luteolysis to ovulation (h)
Single (n = 20)
Double (n = 4)
10.5 0.9 15.9 0.6 1.29 0.11a 102 5
10.8 1.5 14.8 0.6 0.93 0.09b 103 20
None of the parameters were significantly different between single and double ovulating cows. (a,bP = 0.07).
Fig. 2. Plasma estradiol concentrations around estrus in cows exhibiting single (open symbols; n = 20) or double (closed symbols; n = 4) ovulation.
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Table 5 Weight of individual corpora lutea in double ovulating cows slaughtered on day 5 (n = 9) or day 8 (n = 5) of the cycle Day of slaughter
Weight of corpus luteum 1 (g)
Weight of corpus luteum 2 (g)
Mean weight difference (g)
Range of difference (g)
5 8
1.73 0.24 4.24 0.33a
1.30 0.18 2.62 0.39b
0.43 0.16 1.62 0.45
0.0–1.6 0.1–3.1
Within days
a,b
P < 0.01.
These studies have suggested that reduced steroidogenisis feedback leads to elevated gonadotrophin secretion and double ovulations. However, in the present study, in non-lactating animals, we found no difference in circulating progesterone concentrations or in luteal progesterone content or production in animals exhibiting double ovulations. Furthermore, as individual corpora lutea in double ovulating animals were approximately half the weight of corpora lutea in single ovulating animals, total weight of luteal tissue as well as steroidogenic activity of that tissue were also similar in single and double ovulating animals. Thus, reduced peripheral steroid concentrations do no appear to be a prerequisite of double ovulation in cattle. While the number of double ovulating animals in which we had data was small (n = 4), there was no difference in ovulatory follicle diameter between single and double ovulating animals, though preovulatory growth rate tended to be higher (P = 0.07) in single ovulating animals. However, while another small study in a population of cows selected for increased twinning rate [20] found no difference in the size of the two ovulating follicles, it did find that single ovulating follicle diameter (18.4 mm) was larger than double ovulating follicle diameter (13.0–15.5 mm). In the present study the weights of the two corpora lutea were different on day 8 but not on day 5. This finding, together with the observation of no difference in size of the ovulatory follicle in double ovulating cows suggests that the difference of day 8 may result from differential corpus luteum development rather than from differences in the size of the follicle from which the luteal structures developed. The practical importance of information on double ovulation in end of lactation animals is probably quite limited. However, the observation that these animals do exhibit a high incidence of this condition is important as it focuses attention on the need to reassess attitudes to the aetiology of the condition rather than simply blaming high milk yield, which is clearly not the key causal factor. In conclusion, we have found a relatively high incidence of double ovulation in non-lactating dairy cows associated with no difference in total luteal
weight, luteal progesterone production or circulating concentrations of progesterone. Acknowledgements We are grateful to MP Green and LM Hicking for carrying out original studies included in the present analysis and to SJ Mann and AJ Hammond for technical assistance. The original studies were financially supported by the Department for the Environment Food and Rural Affairs, the Milk Development Council, the BBSRC and Intervet, partly under the Link Sustainable Livestock Production Programme. References [1] Mann GE, Lamming GE, Robinson RS, Wathes DC. The regulation of interferon-tau production and uterine hormone receptors during early pregnancy. J Reprod Fertil 1999;(Suppl. 54):317–28. [2] Wiltbank MC, Fricke PM, Sangritasvong S, Sartori R, Ginther OJ. Mechanisms that prevent and produce double ovulations in dairy cattle. J Dairy Sci 2000;83:2998–3007. [3] Kinsel ML, Marsh WE, Ruegg PL, Etherington WG. Risk factors for twinning in dairy cows. J Dairy Sci 1998;81:989–93. [4] Lopez-Gatius F, Lopez-Bejar M, Fenech M, Hunter RHF. Ovulation failure and double ovulation in dairy cattle: risk factors and effects. Theriogenology 2005;63:1298–307. [5] Fricke PM, Wiltbank MC. Effect of milk production on the incidence of double ovulation in dairy cows. Theriogenology 1999;52:1133–43. [6] Lopez H, Caraviello DZ, Satter LD, Fricke PM, Wiltbank MC. Relationship between level of milk production and multiple ovulations in lactating dairy cows. J Dairy Sci 2005;88:2783–93. [7] Lopez H, Sartori R, Wiltbank MC. Reproductive hormones and follicular growth during development of single or multiple dominant follicles in cattle. Biol Reprod 2005;72:788–95. [8] Mann GE, Lamming GE. Relationship between the maternal endocrine environment, early embryo development and the inhibition of the luteolytic mechanism in the cow. Reproduction 2001;121:175–80. [9] Green MP, Hunter MG, Mann GE. Relationships between maternal hormone secretion and embryo development on day 5 of pregnancy in dairy cows. Anim Reprod Sci 2005;88:179–89. [10] Hicking LM, Flint APF, Mann GE. Administration of human chorionic gonadotrophin (hCG) or gonadotrophin releasing hormone (GnRH) analogue at day 5 after estrus and plasma progesterone in the cow. J Anim Sci 2002;80(1):397 [abstract]. [11] Robinson RS, Hammond AJ, Hunter MG, Mann GE. The induction of a delayed post-ovulatory progesterone rise in dairy
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[12]
[13]
[14]
[15] [16]
cows: a novel model. Domest Anim Endocrinol 2005;28: 285–95. Law AS, Baxter G, Logue DN, O’Shea T, Webb R. Evidence for the action of bovine follicular fluid factor(s) other than inhibin in suppressing follicular development and delaying estrus in heifers. J Reprod Fertil 1992;96:603–16. Mann GE, Lamming GE, Fray MD. Plasma oestradiol during early pregnancy in the cow and the effects of treatment with busrelin. Anim Reprod Sci 1995;37:121–31. Tsang PCW, Walton JS, Hansel W. Oxytocin-specific RNA, oxytocin and progesterone concentrations in corpora lutea of heifers treated with oxytocin. J Reprod Fertil 1990;89:77–84. Kidder HE, Barrett GR, Casida LE. A study of ovulations in six families of Holstein-Friesians. J Dairy Sci 1952;35:436–44. Labhsetwar AP, Tyler WJ, Casida LE. Analysis of variation in some factors affecting multiple ovulations in Holstein cattle. J Dairy Sci 1963;46:840–2.
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[17] Santos JE, Huber JT, Theurer CB, Nussio CB, Nussio LG, Tarazon M, et al. Effects of grain processing and bovine somatotropin on metabolism and ovarian activity of dairy cows during early lactation. J Dairy Sci 2000;83: 1004–15. [18] Sartori R, Haughian JM, Shaver RD, Rosa GJ, Wiltbank MC. Comparison of ovarian function and circulating steroids in estrous cycles of Holstein heifers and lactating cows. J Dairy Sci 2004;87:905–20. [19] Lopez-Gatius F, Yaniz JL, Santolaria P, Murugavel K, Guijarro R, Calvo E, et al. Reproductive performance of lactating dairy cows treated with cloprostenol at the time of insemination. Theriogenology 2004;62:677–89. [20] Echternkamp SE. 2000. Endocrinology of increased ovarian folliculogenesis in cattle selected for twin births. Proceedings of the American Society of Animal Science, 1999. Available at: http://www.asas.org/JAS/symposia/proceedings/0935.pdf.
Corpus luteum size and function following single and double ovulations in non-lactating dairy cows G.E. Mann *, R.S. Robinson, M.G. Hunter University of Nottingham, School of Biosciences, Division of Animal Physiology, Sutton Bonington Campus, Loughborough, LE12 5RD, UK Received 20 September 2006; received in revised form 15 January 2007; accepted 15 January 2007
Abstract Data was collated from a number of studies on various aspects of luteal function in non-lactating dairy cows to allow comparisons to be made between single and double ovulating animals. In these studies, estrous cycles had been synchronized and animals slaughtered on day 5 or 8. The overall incidence of double ovulations was 28.3%. Double ovulation was associated with smaller individual corpora lutea but no difference in total weight of luteal tissue or any aspect of luteal tissue function or plasma concentrations of progesterone. Furthermore, in a sub set of animals, there was no difference in preovulatory follicle characteristics or plasma concentrations of estradiol around ovulation. These results demonstrated a high incidence of double ovulation in nonlactating cows that had no influence on circulating progesterone concentrations. # 2007 Elsevier Inc. All rights reserved. Keywords: Cow; Progesterone; Corpus luteum; Ovulation
1. Introduction Numerous studies have identified the importance of postovulatory progesterone secretion from the developing corpus luteum on the outcome of early pregnancy [1]. While cows are primarily a monovular species and progesterone is normally the product of a single corpus luteum, double ovulations are not uncommon, resulting in two corpora lutea contributing to progesterone secretion. A number of studies in dairy cows have reported twinning rates of between 2.4 and 5.8% [2]. A wide range of factors have been linked to twinning in dairy cows including increasing parity, increased or
* Corresponding author. Tel.: +44 1159 516326; fax: +44 1159 516302. E-mail address: [email protected] (G.E. Mann). 0093-691X/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2007.01.011
decreased milk yield, season, genetics, use of reproductive hormones or antibiotics, ovarian cysts and days open [2–4]. In studies in which ovulation rate has been measured directly, typical reported incidences of double ovulation range from around 5–10% in 1st parity, 10– 15% in 2nd parity and 20–25% in 3rd and subsequent parity cows [4,5]. A recent study [6] reported increased total luteal volume but reduced circulating progesterone 7 days after ovulation in double compared with single ovulating cows. Furthermore, another study [7] also reported lower circulating progesterone concentrations during development of multiple dominant follicles. In view of the importance of progesterone during early pregnancy [8] this association between reduced progesterone and multiple ovulation suggests a potential detrimental effect on pregnancy rate. The aim of this study was to determine the effect of double ovulation compared to single on luteal
G.E. Mann et al. / Theriogenology 67 (2007) 1256–1261
characteristics and plasma progesterone concentrations in non-lactating multiparous Holstein-Friesian cows. 2. Materials and methods In this paper, data from a series of previously published studies [9–11] has been collated and reanalysed to permit comparisons to be made between luteal characteristics and circulating progesterone concentrations in cows exhibiting single or double ovulations. All studies were carried out in multiparous Holstein-Friesian cows obtained from local farms at the end of lactation, and were conducted in accordance with the Animals (Scientific Procedures) Act 1986. The mean age of the animals was 5.5 0.3 years and ranged from 2.4 to 11.0 years. In all cases estrous cycles were synchronized prior to slaughter on day 5 or 8 following estrus.
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cannulated to allow collection of blood samples every 8 h throughout the study for progesterone and estradiol analysis. All samples were collected into heparinized tubes, centrifuged at 1500 g for 10 min and the plasma samples were stored at 20 8C until analysis. 2.1.3. Study 3 In this study the estrous cycles of 12 cows were synchronized by two intramuscular injections of 50 mg of the prostaglandin F2a analogue, cloprostenol (Estrumate) administered 12 days apart. Prior to the second cloprostenol injection, a jugular vein of each cow was cannulated to allow collection of blood samples at 8 h intervals. Samples were centrifuged for 10 min at 1500 g and plasma stored at 20 8C for subsequent hormone analysis. Cows were slaughtered on day 8 of the cycle. 2.2. Assessment of corpora lutea
2.1. Experimental animals 2.1.1. Study 1 In this study the estrous cycles of 17 cows were synchronized by two i.m. injections of 50 mg of the prostaglandin F2a analogue, cloprostenol (Estrumate; Schering-Plough Animal Health Welwyn Garden City, UK) administered 12 days apart. On the morning of day 5 following the day of onset of estrus (day 0), a single blood sample (10 ml) was collected by jugular venepunture, centrifuged (1500 g for 10 min) and plasma collected and stored at 20 8C for subsequent analysis. Cows were then transported to a local slaughter facility where they were killed and the reproductive tract was recovered. Ovaries were examined to confirm the appropriate stage of the cycle and corpora lutea collected and snap frozen in liquid nitrogen. 2.1.2. Study 2 In this study the estrous cycles of 24 cows were initially synchronized by inserting a CIDR device (Pfizer Animal Health, Tadworth, UK) for 10 days and injecting 50 mg of the PGF2a analogue, cloprostenol i.m. (Estrumate) on the day of CIDR withdrawal with a second 50 mg i.m. injection 10–15 days later. In these cows, ovaries were scanned daily by transrectal ultrasonography using a Sonovet 6000 ultrasound scanner with a 7.5 MHz linear array transducer (BCF Technology Ltd., Livingstone, UK) from 3 days prior to the second cloprostenol injection until slaughter on day 5 or 8 of the subsequent cycle. Prior to the second cloprostenol injection, a jugular vein of each cow was
In all studies, the number of corpora lutea was noted and all corpora lutea were dissected free of connective tissue, drained of any fluid and weighed. Tissue was then snap frozen and stored at 80 8C for subsequent determination of progesterone content. In studies 1 and 3, luteal tissue from all animals was finely minced using scalpel blades and washed in phosphate buffered saline by centrifugation at 1500 g for 3 min at 4 8C and the supernatant discarded. Aliquots of approximately 25 mg of tissue ( 2mg) were placed into tubes containing 1 ml of Medium 199 containing 10 mM NaHCO3 1-1, Earle’s salts, 0.068 mM L-glutamine and 25 mM HEPES (Sigma– Aldrich Ltd., Poole, Dorset, UK). Luteal tissue was incubated in control media or media supplemented with 100 ng/ml bovine LH (AFP11743 B biopotency 1.06x oLH NIDDK-I-2). Tissue was incubated for 30 min at 38 8C in a shaking water bath at 70 strokes/min. Following incubation, the supernatant was aspirated and both medium and luteal tissues were snap frozen and stored at 80 8C. In addition, minced tissue was collected prior to incubation to determine initial progesterone content. 2.3. Hormone analysis Plasma progesterone concentrations were measured by radioimmunoassay [12] with a limit of sensitivity of 0.3 ng/ml and the intra- and inter-assay coefficients of variation of 7.3 and 12.6%, respectively. Plasma estradiol concentrations were measured in plasma after diethyl ether extraction using a radioimmunoassay kit
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G.E. Mann et al. / Theriogenology 67 (2007) 1256–1261
Table 1 Distribution of single and double ovulating cows across studies and day of slaughter Study
Day of slaughter
Single ovulation (n)
1 2
5 5 8
11 8 12
3
8
Overall
Double ovulation (n) 6 3 1
Incidence (%) 35.3 16.7
7
5
41.7
38
15
28.3
Table 2 Luteal characteristics and plasma progesterone concentration in single and double ovulating cows slaughtered on day 5 and 8 of the estrous cycle Day 5
Day 8
Single (n = 19) Corpus luteum weight (g) Total luteal weight (g) Tissue progesterone (ng/mg) Plasma progesterone (ng/ml) Within days
a,b
P < 0.001; between days
2.9 0.3 2.9 0.3 14.3 1.5 2.0 0.2
a
Double (n = 9) 1.5 0.2 3.0 0.4 13.6 1.4 2.1 0.5
b
Single (n = 19) a,***
6.3 0.5 6.3 0.5 *** 12.8 0.9 6.1 0.5 ***
Double (n = 6) 3.4 0.3b,*** 6.9 0.6 *** 13.7 0.8 5.6 0.7 ***
***
P < 0.001.
(E2 MAIA Serono Diagnostics, Woking, UK) modified for use in the cow [13]. The sensitivity of the assay was 0.5 pg/ml and the intra- and inter-assay coefficients of variation were 13.5 and 13.1%, respectively. Tissue progesterone content was determined following homogenisation and petroleum ether extraction [14] followed by radioimmunoassay as described above. 2.4. Statistical analysis Incidence of double ovulation was analysed by chi-squared test following Yates’s correction. Luteal and follicular characteristics in single and double ovulating animals were compared by analysis of variance with study as a factor. Where repeated progesterone and estradiol concentrations were analysed, repeated sample analysis of variance was undertaken.
summer (28.0%) was not different to that seen during autumn/winter (28.6%). On both day 5 and 8, individual corpora lutea were smaller (P < 0.001) in double ovulating cows though the total weight of luteal tissue was the same as single and double ovulating animals (Table 2). In addition, the progesterone content of luteal tissue was similar in corpora lutea collected from single and double ovulating animals on both day 5 and 8 (Table 2). Thus, the total luteal content of progesterone was also similar between single and double ovulating animal (data not shown). Plasma progesterone concentration on the day of slaughter was also similar between single and double ovulating animals on day 5 and 8 (Table 2). Furthermore,
3. Results The overall incidence of double ovulation across studies was 28.3%. While the incidence was not the same the three studies (Table 1), these differences were not significant. While the incidence of double ovulations was lower in younger (<4.5 years; 3/17–17.6%) than in older (>4.5 years; 12/36–33.3%) cows, this difference was not significant. Furthermore, the mean age of double ovulating cows (5.7 0.6 years) was not different from single ovulating cows (5.4 0.3 years). The incidence of double ovulation during spring/
Fig. 1. Plasma progesterone concentration from day 5 to 8 in single (open circles; n = 19) and double (closed circles; n = 6) ovulating cows slaughtered on day 8.
G.E. Mann et al. / Theriogenology 67 (2007) 1256–1261
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Table 3 In vitro progesterone secretion by luteal tissue collected from single and double ovulating cows slaughtered on day 5 and 8 of the estrous cycle Day 5
Basal tissue progesterone synthesis in vitro (ng/mg) LH stimulated progesterone synthesis in vitro (ng/mg)
Day 8
Single (n = 11)
Double (n = 6)
Single (n = 7)
Double (n = 5)
43.4 5.0 50.8 5.8
43.8 5.2 49.0 4.1
40.0 2.6 49.1 3.7
39.0 2.6 47.9 2.5
LH caused an overall stimulation of progesterone secretion (P < 0.01) but there was no effect of ovulation number (n: number of animals from which tissue was incubated).
in cows slaughtered on day 8, plasma concentrations of progesterone from day 5 to 8 were also similar (Fig. 1). In studies 1 and 3, incubation of luteal tissue from single and double ovulating cows slaughtered on day 5 and 8 revealed no difference in basal LH production. Furthermore, while there was a significant stimulation of progesterone secretion by LH, this response did not differ between single and double ovulating animals (Table 3). In study 2, ultrasound scanning allowed assessment of preovulatory follicle development in single and double ovulating animals. There was no significant difference in size of ovulatory follicle at either luteolysis or ovulation (Table 4). Furthermore, there was no difference in the time from luteolysis to ovulation. However, growth rate of the preovulatory follicle tended (P < 0.07) to be higher in single ovulating cows. Measurement of plasma estradiol concentration around ovulation revealed no differences between single and double ovulating animals (Fig. 2). When the weights of individual corpora lutea in double ovulating animals were compared, on day 5 there was no significant difference between the two corpora lutea while on day 8 the smaller corpora lutea were significantly lighter than the larger corpora lutea (Table 5). However, on both days there was a wide spread in the range of differences with some cows having 2 corpora lutea of the same size and others markedly different corpora lutea. This relationship was unaffected by the occurrence of unilateral (n = 10) or bilateral (n = 5) ovulation.
4. Discussion Early studies evaluating the ovulation rate by rectal palpation of number of corpora lutea reported multiple ovulation rates ranging from 5 to 13% [15,16]. More recent studies in high yielding dairy cows have reported rates of 10–39% [4,5,17,18,19] and high milk yield has been suggested as the principle cause of double ovulation in modern dairy cows [3,5]. However, in the present study we have reported a relatively high incidence of double ovulation (28.3%) in non-lactating dairy cows. This suggests that while high milk yield may be involved in the aetiology double ovulation, is not a prerequisite for a relatively high incidence of double ovulation in Holstein-Friesian dairy cows and is unlikely to be a major causal factor. Another causal factor heavily implicated in double ovulation is parity [4,5] and it has been reported that parity determines the incidence of double ovulation independently of milk yield [4]. In the present study, while animal numbers were insufficient to allow robust comparison, the incident of double ovulation in younger cows was lower than in older cows. Other studies, again in lactating animals, have reported associations between double ovulation and reduced circulating concentrations of progesterone [7].
Table 4 Ovulatory characteristics in cows exhibiting single (n = 20) or double (n = 4) ovulation
Follicle diameter at luteolysis (mm) Follicle diameter at ovulation (mm) Growth rate (mm/24 h) Time from luteolysis to ovulation (h)
Single (n = 20)
Double (n = 4)
10.5 0.9 15.9 0.6 1.29 0.11a 102 5
10.8 1.5 14.8 0.6 0.93 0.09b 103 20
None of the parameters were significantly different between single and double ovulating cows. (a,bP = 0.07).
Fig. 2. Plasma estradiol concentrations around estrus in cows exhibiting single (open symbols; n = 20) or double (closed symbols; n = 4) ovulation.
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Table 5 Weight of individual corpora lutea in double ovulating cows slaughtered on day 5 (n = 9) or day 8 (n = 5) of the cycle Day of slaughter
Weight of corpus luteum 1 (g)
Weight of corpus luteum 2 (g)
Mean weight difference (g)
Range of difference (g)
5 8
1.73 0.24 4.24 0.33a
1.30 0.18 2.62 0.39b
0.43 0.16 1.62 0.45
0.0–1.6 0.1–3.1
Within days
a,b
P < 0.01.
These studies have suggested that reduced steroidogenisis feedback leads to elevated gonadotrophin secretion and double ovulations. However, in the present study, in non-lactating animals, we found no difference in circulating progesterone concentrations or in luteal progesterone content or production in animals exhibiting double ovulations. Furthermore, as individual corpora lutea in double ovulating animals were approximately half the weight of corpora lutea in single ovulating animals, total weight of luteal tissue as well as steroidogenic activity of that tissue were also similar in single and double ovulating animals. Thus, reduced peripheral steroid concentrations do no appear to be a prerequisite of double ovulation in cattle. While the number of double ovulating animals in which we had data was small (n = 4), there was no difference in ovulatory follicle diameter between single and double ovulating animals, though preovulatory growth rate tended to be higher (P = 0.07) in single ovulating animals. However, while another small study in a population of cows selected for increased twinning rate [20] found no difference in the size of the two ovulating follicles, it did find that single ovulating follicle diameter (18.4 mm) was larger than double ovulating follicle diameter (13.0–15.5 mm). In the present study the weights of the two corpora lutea were different on day 8 but not on day 5. This finding, together with the observation of no difference in size of the ovulatory follicle in double ovulating cows suggests that the difference of day 8 may result from differential corpus luteum development rather than from differences in the size of the follicle from which the luteal structures developed. The practical importance of information on double ovulation in end of lactation animals is probably quite limited. However, the observation that these animals do exhibit a high incidence of this condition is important as it focuses attention on the need to reassess attitudes to the aetiology of the condition rather than simply blaming high milk yield, which is clearly not the key causal factor. In conclusion, we have found a relatively high incidence of double ovulation in non-lactating dairy cows associated with no difference in total luteal
weight, luteal progesterone production or circulating concentrations of progesterone. Acknowledgements We are grateful to MP Green and LM Hicking for carrying out original studies included in the present analysis and to SJ Mann and AJ Hammond for technical assistance. The original studies were financially supported by the Department for the Environment Food and Rural Affairs, the Milk Development Council, the BBSRC and Intervet, partly under the Link Sustainable Livestock Production Programme. References [1] Mann GE, Lamming GE, Robinson RS, Wathes DC. The regulation of interferon-tau production and uterine hormone receptors during early pregnancy. J Reprod Fertil 1999;(Suppl. 54):317–28. [2] Wiltbank MC, Fricke PM, Sangritasvong S, Sartori R, Ginther OJ. Mechanisms that prevent and produce double ovulations in dairy cattle. J Dairy Sci 2000;83:2998–3007. [3] Kinsel ML, Marsh WE, Ruegg PL, Etherington WG. Risk factors for twinning in dairy cows. J Dairy Sci 1998;81:989–93. [4] Lopez-Gatius F, Lopez-Bejar M, Fenech M, Hunter RHF. Ovulation failure and double ovulation in dairy cattle: risk factors and effects. Theriogenology 2005;63:1298–307. [5] Fricke PM, Wiltbank MC. Effect of milk production on the incidence of double ovulation in dairy cows. Theriogenology 1999;52:1133–43. [6] Lopez H, Caraviello DZ, Satter LD, Fricke PM, Wiltbank MC. Relationship between level of milk production and multiple ovulations in lactating dairy cows. J Dairy Sci 2005;88:2783–93. [7] Lopez H, Sartori R, Wiltbank MC. Reproductive hormones and follicular growth during development of single or multiple dominant follicles in cattle. Biol Reprod 2005;72:788–95. [8] Mann GE, Lamming GE. Relationship between the maternal endocrine environment, early embryo development and the inhibition of the luteolytic mechanism in the cow. Reproduction 2001;121:175–80. [9] Green MP, Hunter MG, Mann GE. Relationships between maternal hormone secretion and embryo development on day 5 of pregnancy in dairy cows. Anim Reprod Sci 2005;88:179–89. [10] Hicking LM, Flint APF, Mann GE. Administration of human chorionic gonadotrophin (hCG) or gonadotrophin releasing hormone (GnRH) analogue at day 5 after estrus and plasma progesterone in the cow. J Anim Sci 2002;80(1):397 [abstract]. [11] Robinson RS, Hammond AJ, Hunter MG, Mann GE. The induction of a delayed post-ovulatory progesterone rise in dairy
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