Reprod Dom Anim 45, 500 504 (2010); doi: 10.1111/j.1439-0531.2008.01277.x ISSN 0936-6768 Comparison in Effect of with Heat Detection Aids and CIDR- in Dairy Heifers M Yusuf 1, T Nakao 1, C Yoshida 1, ST Long 1, S Fujita 1, Y Inayoshi 2 and T Furuya 2 1 Department of Veterinary Medicine, Faculty of Agriculture, Yamaguchi University, Yoshida; 2 Yamaguchi Prefectural Livestock Research Institute, Mine-gun, Mitou-Chou, Yamaguchi, Japan Contents The objective of the present study was to determine whether oestrous detection with the help of oestrous detection aids during the without timed AI protocol is equally effective with the progesterone-combined protocol in dairy heifers. A total of 148 heifers were randomly assigned to one of the two groups. A group of heifers treated with with heat detection aids (n = 72) received GnRH on day 0, prostaglandin F 2a (PGF 2a ) on day 7 and oestradiol benzoate (EB) on day 8, while in controlled internal drug release (CIDR)- group (n = 76), CIDR was included during a period from GnRH to PGF 2a. Heifers were checked for oestrus twice daily, i.e. from 09:00 to 10:00 hours and from 15:00 to 16:00 hours starting on day 2 for group and on day 8 in CIDR- group, and continued up to day 12. KAMAR Ò heat mount detector (KAMAR Ò Inc., Steamboat Springs, CO, USA) and ALL-WEATHER Ò PAINTSTIK Ò (LA-CO Industries Inc., Elk Grove Village, IL, USA) were used as heat detection aids. AI was conducted within 1 h after confirming oestrus in 72 heifers, while 19 animals were transferred with embryo 7 days after oestrus according to the request of the owners. Premature oestrus before PGF 2a injection occurred in 18% of group. Of 13 heifers which showed premature oestrus, six were inseminated and two of them conceived. Oestrus detection rate within 12 days after initiation of the protocols did not differ between the two groups (94% vs 95%). There was no difference in the conception rate after first AI (including heifers that were inseminated before PGF 2a injection) and embryo transfer between with heat detection aids and CIDR- groups (36% vs 44% and 70% vs 56%). It is concluded that the use of heat detection aids to monitor the occurrence of premature oestrus prior to PGF 2a injection in protocol in dairy heifers was equally effective to the inclusion of CIDR. Introduction Synchronization of oestrus in cattle implies the manipulation of the oestrous cycle or induction of oestrus to bring a large percentage of cattle in a herd into oestrus at predetermined time (Odde 1990) and to reduce the need for oestrous detection (DeJarnette et al. 2001). This technique has widely been used as an important tool for increasing AI submission rate in beef and dairy herds (Macmillan and Peterson 1993; Xu and Burton 1999; Lucy et al. 2001). Most commonly used oestrus ovulation synchronization protocols in dairy cows may include Ovsynch timed artificial insemination (TAI) (Pursley et al. 1995; Twagiramungu et al. 1995; Schmitt et al. 1996), (Pancarci et al. 2002) and Select Synch (Twagiramungu et al. 1995; Burke et al. 1996; Tenhagen et al. 2005). It has already been reported that Ovsynch TAI protocols do not work in dairy heifers as much as in cows (Twagiramungu et al. 1995; Pursley et al. 1997). This may be because of the increased odds of administering the first GnRH in the absence of a dominant follicle in heifers (Haughian and Wiltbank 2002), leading to a low follicle turnover success and failure to induce a new follicular wave. As a consequence, corpus luteum (CL) is absent at the day of prostaglandin F 2a (PGF 2a ) injection and oestrus (premature oestrus) and ovulation occurs around the day of PGF 2a injection (DeJarnette et al. 2001). To overcome the disadvantages of these protocols for heifers, progestins have often been administered during a period from the first GnRH to PGF 2a (Peeler et al. 2004; Ambrose et al. 2005; Cavalieri et al. 2006). The occurrence of oestrus before or at around the day of PGF 2a injection was substantially reduced by the combined use of progestin and as a consequence oestrous induction rate within 5 days after PGF 2a has been increased. However, if oestrus occurring before PGF 2a is accurately detected with the use of oestrous detection aids and AI is conducted based on the detection of oestrus, the use of progestin can be avoided, without causing a decrease in AI submission rate during a treatment period. The aim of this study was, therefore, to determine whether oestrous detection with the help of oestrous detection aids during the protocol is equally effective as with the progesterone-combined protocol in dairy heifers. Although is a protocol with timed AI, in the present study, the authors modified the protocol; AI was conducted after detection of oestrus, because oestrus observation was carried out as a routine in this herd. Materials and Methods Heifer Raising and housing This study was conducted in Yamaguchi Prefectural Heifer Raising Breeding Farm, Yamaguchi, Japan. Heifers were brought here from a number of dairy farms within the prefecture at 1 3 months of age have being kept until the animals become pregnant. The pregnant heifers were returned to each farm a few month before due date. The herd was kept on the free barn. The concrete floor was covered with sawdust. Heifers were submitted to AI after the age of 12 months with height of 125 cm or more and body weight of
Effect of with Heat Detection Aids and CIDR- 501 350 kg or more. The animals were checked for oestrus twice daily, from 09:00 to 10:00 hours and from 15:00 to 16:00 hours, with the help of oestrous detection aid (ALL-WEATHER Ò PAINTSTIK Ò ) and AI was conducted within 1 h after confirming oestrus based on the change of the tail paint and palpation of the reproductive tract per rectum. This means that heifers, which showed oestrus during a period from 16:00 to 09:00 hours on the following day were inseminated between 09:00 and 10:00 hours, and those in oestrus from 10:00 to 15:00 hours were bred between 15:00 and 16:00 hours. In some heifers, embryos from Japanese Black beef cattle were transferred in utero 7 days after oestrus depending on the request of the owners. Heifers that had not been detected oestrus for more than 1 month by the routine observation were assigned to the experiment for oestrous synchronization. The herd was visited at 1 month interval, and a group of four to 10 heifers were referred to the experiment. They were divided into two treated groups. with heat detection aids and controlled internal drug release (CIDR)- A total of 148 Holstein heifers at 12 16 months of age were enrolled in the study during a period from August 2005 to March 2007. Of the 148 heifers, 78 were due to be bred by AI and 70 were due to be embryo transferred according to the owners request. The heifers were randomly assigned to one of the two treated groups at the beginning of treatment. Seventy-two heifers in with heat detection aids group, received 100 lg of GnRH-analogue (fertirelin acetate, Conceral Ò, Schering-Plough Animal Health, Tokyo, Japan) i.m. at the beginning of the treatment, followed by 500 lg PGF 2a -analogue (Cloprostenol, Resipron Ò -C, ASKA Pharmaceutical Co., Ltd, Tokyo, Japan) i.m. 7 days later and 0.5 mg EB (Kawasaki-Mitaka Pharmaceuticals Co., Ltd, Tokyo, Japan) i.m. at the day after PGF 2a treatment. In CIDR- group (n = 76), heifers were treated with GnRH and PGF 2a at an interval of 7 days, and EB at the day after PGF 2a treatment. At the time of GnRH administration, a CIDR (EAZI-BREED CIDR Ò, Livestock Improvement Association of Japan, Tokyo, Japan) was inserted into the vagina. The CIDR was removed on the day of PGF 2a administration (Fig. 1). Two types of heat detection devices, KAMAR Ò heat mount detector (HMD) and ALL-WEATHER Ò PAINTSTIK Ò (tail paint) were used. Heifers in with heat detection aids and CIDR- groups were applied with one of the two devices on the day of GnRH and PGF 2a treatments, respectively (Fig. 1). Oestrous signs or the change of the oestrous detection devices were checked twice daily as a routine. The animals showing positive response of the devices, wearing off of tail paint or 50% or more reaction of HMD with red colour, were palpated per rectum. Those showing clear uterine contraction were confirmed to have been in oestrus. Heifers assigned for AI were inseminated artificially with frozen thawed semen from proven sires within 1 h after confirming oestrus, while in the heifers assigned for embryo transfer (ET), an Fig. 1. Protocols of oestrous synchronization in dairy heifers; upper figure for with heat detection aids, lower figure for CIDR- embryo of grade A or B of fresh embryo or grade A of frozen embryo was transferred non-surgically into the uterus 7 days after oestrus was detected. Blood sampling and hormone assay Blood samples were collected from all heifers on the days of GnRH treatment, PGF 2a treatment and EB treatment via coccygeal venipuncture into evacuated heparinized vacuum tubes (VENOJECT Ò II TERUMO, Terumo Corporation, Tokyo, Japan). After collection, samples were kept at 4 C and were centrifuged within 2 h at 1500 g for 15 min to collect plasma. The plasma was stored frozen at )20 C until assayed for progesterone concentrations by enzyme immunoassay (Isobe and Nakao 2003). The intra-assay coefficient of variation was 12.8%. The inter-assay coefficients of variation were 7.8% (8.8 ng ml) and 19.7% (0.5 ng ml), respectively. Reproductive performance AI submission rate or ET submission rate was defined as the number of heifers inseminated or transferred with an embryo divided by the number of heifers to be inseminated or to receive an embryo via ET. Conception rate after AI or ET was defined as the number of heifers conceiving divided by total number of heifers inseminated or received an embryo. Pregnancy was checked by trans-rectal palpation 40 days after AI or 33 days after ET or later. Pregnancy rate after AI or ET was defined as the number of heifers conceiving divided by total number of heifers to be inseminated or to receive an embryo. Response of ovaries after the synchronization treatments based on plasma progesterone concentrations Effect of the treatment was also evaluated based on plasma progesterone concentrations on the days of
502 M Yusuf, T Nakao, C Yoshida, ST Long, S Fujita, Y Inayoshi and T Furuya GnRH treatment (day 0), PGF 2a treatment (day 7), and EB treatment (day 8). Progesterone concentrations below 1.0 ng ml were considered low and indicative of the absence of CL, while progesterone concentrations of 1.0 ng ml or higher were considered as an indication of functional CL and were referred as high (Cordoba and Fricke 2002; Rivera et al. 2004). Heifers showing progesterone response high to high to low (H fi H fi L) and low to high to low (L fi H fi L) at the day of GnRH treatment, 7 days after GnRH, and 1 day after PGF 2a were shown as positive responses to GnRH and PGF 2a treatments, presence of CL 7 days after GnRH and regression of CL after PGF 2a. Statistical analysis Statistical analysis was carried out using the statistical package SPSS 12.0 for windows (SPSS Inc., Chicago, IL, USA). Differences between protocols in the percentages of heifers coming into oestrus before PGF 2a administration were analysed by Fisher s exact test. Percentages of heifers coming into oestrus within 5 days after PGF 2a administration, total number of heifers coming into oestrus, AI submission rate, ET submission rate, conception rate after AI or ET, and pregnancy rate after AI or ET were analysed by Chi-square test. The level of significance was set at p 0.05. Results Table 1 shows the effect of with heat detection aids and CIDR- on heat detection rates and reproductive performance in heifers. Of 72 heifers in with heat detection aids group, 13 Table 1. Effects of with heat detection device and CIDR- on heat detection rates and reproductive performance in dairy heifers with heat detection device CIDR- p-value Total No. of heifers treated 72 76 No. of heifers coming into 13 (18) 0 (0) <0.01 oestrus before PGF 2a administration (%) (a) No. of heifers coming into oestrus 55 (76) 72 (95) 0.146 within 5 days after PGF 2a administration (%) (b) a + b 68 (94) 72 (95) 0.884 No. of heifers to be inseminated 36 42 AI submission rate a (%) 33 (92) 39 (93) 0.941 Conception rate b (%) 12 (36) 17 (44) 0.371 Pregnancy rate c (%) 12 (33) 17 (41) 0.352 No. of heifers to receive an 36 34 embryo via ET ET submission rate d (%) 10 (28) 9 (27) 0.893 Conception rate e (%) 7 (70) 5 (56) 0.212 Pregnancy rate f (%) 7 (19) 5 (15) 0.493 a Number of heifers inseminated number of heifers to be inseminated. b Number of heifers pregnant number of heifers inseminated. c Number of heifers pregnant number of heifers to be inseminated. d Number of heifers received an embryo via ET number of heifers yet to receive an embryo. e Number of heifers pregnant number of heifers received an embryo. f Number of heifers pregnant number of heifers yet to receive an embryo. (18%) heifers showed premature oestrus before PGF 2a administration. Of 13 heifers which were showing premature oestrus, six heifers were inseminated and two of them conceived. On the other hand, no heifer in CIDR- group was detected with oestrus before PGF 2a injection. Oestrous detection rate within 5 days after PGF 2a administration was 76% in with heat detection aids group, while it was 95% in CIDR- group. The percentages of heifers coming into oestrus during a 12-day period from GnRH treatment to 5 days after PGF 2a injection in both groups were not significantly different (94% vs 95%). There was no significant difference in oestrus detection efficacy of the two oestrus detection devices. AI submission rate, conception rate and pregnancy rate did not differ between the two groups (92% vs 93%, 36% vs 44% and 33% vs 42%). Conception rate in the two groups after ET did not differ (70% vs 56%). Plasma progesterone concentrations (Fig. 2) showed that 50 (69%) of 72 heifers in with heat detection aids group and 54 (71%) of 76 heifers in CIDR- group had functional CL at the beginning of treatment. Of 72 heifers in with heat detection aids group, 43 (60%) heifers showed ovulation and CL formation after GnRH and regression of CL after PGF 2a administration. Sixteen (22%) heifers had negative responses to GnRH and or PGF 2a. Thirteen other heifers (18%) showed premature oestrus before PGF 2a administration and, therefore, no more blood sampling and treatment was conducted. Likewise, in CIDR- group, 65 (86%) of 76 heifers showed positive response to GnRH and PGF 2a and eleven (15%) had negative response. Discussion In this study, oestrous induction rate in with heat detection aids within 5 days after PGF 2a was 76%. The occurrence of premature oestrus before PGF 2a injection was 18%. With combined use of CIDR during a period from GnRH treatment to PGF 2a injection, no animal showed premature oestrus and as a consequence, oestrous induction rate within 5 days after PGF 2a was improved up to 95%. However, when 13 animals that showed premature oestrus were included, oestrous detection rate in with heat detection aids group was 94%, similar with the oestrous detection rate in CIDR- group. Oestrous induction rate in with heat detection aids and CIDR- groups were comparable with the results of previous studies (Schmitt et al. 1996; Lucy et al. 2001; Tenhagen et al. 2005). No difference in the efficacy of the two oestrous detection aids was shown in this study. The use of paintstik costing approximately 1.4 US dollars for one case seems to be more economical than the use of HMD costing approximately 3.8 US dollars in our circumstances. AI submission rates in with heat detection aids group during a 12-day period from GnRH treatment to 5 days after PGF 2a injection and CIDR- group within 5 days after PGF 2a injection were 92% and 93%, respectively, which correspond with previous reports (Schmitt et al. 1996; Lucy et al. 2001;
Effect of with Heat Detection Aids and CIDR- 503 Fig. 2. Patterns of response of plasma progesterone in heifers after GnRH and PGF 2a in with heat detection aids group and after GnRH and CIDR, PGF 2a in CIDR- group Tenhagen et al. 2005). AI submission rates in the two groups were almost equal. This indicates that the use of oestrous detection aids during a period from GnRH treatment to PGF 2a injection is useful to improve AI submission rate in heifers. Occurrence of premature oestrus during oestrous synchronization period in with heat detection aids was 18%. The percentage of heifers showing premature oestrus in this experiment was almost same as those obtained in the previous studies in dairy heifers (Rivera et al. 2004) and in dairy cows (DeJarnette et al. 2001; Kim et al. 2003) following Ovsynch protocol. DeJarnette et al. (2001) also reported that 20% of GnRH-treated cows displayed premature oestrus and necessitate oestrous detection during this period if maximal pregnancy rates are to be achieved. Detecting the occurrence of premature oestrus after GnRH treatment with aid of heat detection devices is shown to be as effective as the combined use of CIDR in protocol in heifers. However, the goal of protocol is to synchronize oestrus and ovulation after EB injection to allow us to do timed AI. Addition of CIDR improved oestrus detection rate after EB administration in this study. Percentage of heifers in CIDR- group showing positive response of plasma progesterone to GnRH and PGF 2a tended to be higher than the percentage in with heat detection aids group. Higher percentage of heifers with positive response of plasma progesterone in CIDR- group was partly because of CIDR insertion. In this study, the heifers were at random stages of the oestrous cycle at the beginning of the treatment. Pursley et al. (1995) earlier reported that only 54% of heifers responded to GnRH with ovulation and CL formation, compared with 90% in lactating cows. It was also reported that initiation of the protocol at day 15 of the oestrous cycle caused premature ovulation in heifers (Moreira et al. 2000). Heifers may have an increased odd of initiation of the protocol when they have no dominant follicle to respond to GnRH (Haughian and Wiltbank 2002) and this may cause the absence of CL at PGF 2a injection and premature oestrus (DeJarnette et al. 2001). In the present study, the use of heat detection aids during protocol to detect premature oestrus improved AI submission rate as high as in CIDR- group. It is suggested that under the circumstances where oestrous detection is conducted as a routine, the detection of premature oestrus with the help of heat detection aids after the initial treatment for is a good alternative to CIDR inserts. In conclusion, the use of heat detection aids to detect the occurrence of premature oestrus prior to PGF 2a injection in protocol in dairy heifers was equally effective to the inclusion of CIDR in AI submission rate within 12 days after initiation of the protocols. Acknowledgements We thank Schering-Plough Animal Health (Tokyo, Japan) for GnRH and PGF 2a, Kawasaki-Mitaka Pharmaceuticals Co., Ltd. (Tokyo, Japan) for oestradiol benzoate, and Livestock Improvement Association of Japan for CIDR. First author is supported by the Monbukagakusho Scholarship of Japan. Author contributions M Yusuf, T Nakao and C Yoshida had contributed in designing of the study, collecting data, analysing data and drafting paper. ST Long, S Fujita, Y Inayoshi and T Furuya had contributed in the study for collecting data and analysing the data. References Ambrose JD, Kastelic JP, Rajamahendran R, Aali M, Dinn N, 2005: Progesterone (CIDR)-based timed AI protocols using GnRH, porcine LH or estradiol cypionate for dairy heifers: ovarian and endocrine responses and pregnancy rates. Theriogenology 64, 1457 1474. Burke JM, De La Sota RL, Risco CA, Staples CR, Schmitt Eˇ JP, Thatcher WW, 1996: Evaluation of timed insemination using a gonadotropin-releasing hormone agonist in lactating dairy cows. J Dairy Sci 79, 1385 1393. Cavalieri J, Hepworth G, Smart VM, Ryan M, Macmillan KL, 2006: Reproductive performance of lactating dairy cows and heifers resynchronized for a second insemination with an intravaginal progesterone-releasing aid for 7 or 8 d with estradiol benzoate injected at the time of aid insertion and 24 h after removal. Theriogenology 67, 824 834. Cordoba MC, Fricke PM, 2002: Initiation of the breeding season in a grazing-based dairy by synchronization of ovulation. J Dairy Sci 85, 1752 1763. DeJarnette JM, Salverson RR, Marshall CE, 2001: Incidence of premature estrus in lactating dairy cows and conception
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