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Comparison between two estradiol-progesterone based protocols for timed artificial insemination in blocks in lactating Nelore cows
Accepted Manuscript Title: Comparison between two estradiol-progesterone based protocols for timed artificial insemination in blocks in lactating Nelore cows Authors: L.F.M. Pfeifer, N.A. Castro, P.M.A. Neves, J.P. Cestaro, A. Schneider PII: DOI: Reference:
S0378-4320(16)30586-3 http://dx.doi.org/doi:10.1016/j.anireprosci.2017.03.025 ANIREP 5577
To appear in:
Animal Reproduction Science
Received date: Accepted date:
25-10-2016 31-3-2017
Please cite this article as: Pfeifer, L.F.M., Castro, N.A., Neves, P.M.A., Cestaro, J.P., Schneider, A., Comparison between two estradiol-progesterone based protocols for timed artificial insemination in blocks in lactating Nelore cows.Animal Reproduction Science http://dx.doi.org/10.1016/j.anireprosci.2017.03.025 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Comparison between two estradiol-progesterone based protocols for timed artificial insemination in blocks in lactating Nelore cows L.F.M. Pfeifera*, N.A. Castrob, P.M.A. Nevesc, J.P. Cestaroc, A. Schneiderb Embrapa, Brazilian Agricultural Research Corporation, Porto Velho, Rondônia, Brazil
a
b
Departamento de Nutrição, Universidade Federal de Pelotas, Pelotas, Rio Grande do
Sul, Brazil FIMCA, Faculdades Integradas Aparício Carvalho, Porto Velho, Rondônia, Brazil
c
*Corresponding Author: Luiz Francisco Machado Pfeifer; Embrapa Rondônia, BR 364 Km 5,5 - Zona Rural, Caixa postal: 127 CEP: 76815-800, Porto Velho - Rondônia – Brazil; Business phone: 01155 69 3901-2510; E-mail: [email protected]
1
ABSTRACT The aim of this study was to compare the use of artificial insemination in time blocks (Artificial Insemination Blocks, AIB) using an 8 and 9 d estradiol-progesterone based protocol. In this experiment, lactating Nelore cows (n = 253) were subjected to two estradiol-progesterone based TAI protocols. On the morning of Day 10 (8d group, n = 124) or Day 11 (9d group, n = 129), cows were examined by ultrasonography to evaluate the diameter of the preovulatory follicle and were inseminated once at one of the following time points, according to the diameter of the pre-ovulatory follicle (POF): Block 0 (POF ≥ 15 mm, TAI 0 h after conventional TAI), Block 1 (POF 13.0 to 14.9 mm, TAI 6 h later), Block 2 (POF 10.1 to 12.9 mm, TAI 24 h later), and Block 3 (POF ≤ 10.0 mm, TAI 30 h later). The pregnancy per AI (P/AI) did not differ between 8d and 9d groups (P > 0.05). Considering only multiparous cows, however, P/AI tended to be greater in the 8d (64.1%) than in the 9d group (49.3%; P = 0.08). Cows from the 9d group tended to have a larger POF than cows from the 8d group (P = 0.07). In conclusion, these results provide evidence that there is no difference between 8d or 9d protocols when using the AIB technique. Use of the 8d estradiol-progesterone based protocol, however, tended to increase pregnancy in multiparous cows.
Keywords: Cattle; Follicular diameter; Ovulation; Ultrasonography
1. Introduction Artificial insemination (AI) is one of the main techniques used worldwide to disseminate desirable genetics among beef and dairy herds (Bo et al., 2016) and the development of timed AI (TAI) protocols contributed to the widespread use of the AI technique. In Brazil, semen commercialization increased 300% between 2000 and 2011 due to use of TAI (ASBIA, 2011). Data collected from the 2015 breeding season suggest
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that the number of cattle involved in TAI programs was about 10,000,000 in Brazilian commercial herds (Bo et al., 2016). Since the first report of the Ovsynch (Pursley et al., 1995) and estradiolprogesterone based protocols (Bo et al., 1994), estrous synchronization programs have been modified to improve fertility response of inseminated cows. However, only small improvements in the fertility of cows occurred when TAI protocols were used (Bo et al., 2016; Day, 2015; Meneghetti et al., 2009; Sa Filho et al., 2010; Sa Filho et al., 2013; Whittier et al., 2013). The most used protocol for Nelore cows maintained in tropical areas consists of an intravaginal progesterone insert, estradiol benzoate (EB) to induce synchronous ovarian follicular wave emergence, prostaglandin F2a analogues (PGFs) to induce luteolysis, and an ovulatory stimulus using an ester of estradiol (benzoate or cypionate). This estradiol-based protocol and its modifications provided satisfactory fertility in commercial Brazilian herds, independent of breed (B. indicus, B. taurus, or crossbreds). The 8d and 9d estradiol-progesterone based protocols were described previously by Baruselli et al. (2004) and Meneghetti et al. (2009), respectively, and are recognized as the most used protocols for TAI in lactating Nelore cows in Brazil. Currently, such TAI protocols result in pregnancy rates per AI (P/AI) that range from 40% to 60%, therefore, new alternatives to increase fertility of cows submitted to TAI programs are necessary. Recently, a technique known as AI in time blocks (Artificial Insemination Blocks, AIB) was developed in attempts to increase fertility with use of estradiolprogesterone based protocols for TAI (Pfeifer et al., 2015). The use of AIB protocols resulted in a P/AI of 60% in lactating Nelore cows. Because the AIB technique was developed to be used in 8d estradiol-progesterone based protocol, there are questions if the same blocking system for time of AI would be effective with use of 9d protocols. Based on these considerations, the objective of the present study was to compare the P/AI for cows inseminated in time blocks based on size of the largest follicle using an 8d and 9d protocol.
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2. Materials and methods The Committee for Ethics in Animal Experimentation from the Embrapa approved all of the procedures performed in the experiments described in this manuscript (Protocol F02.2014). 2.1. Animal treatments Lactating beef cows (B. indicus, Nelore; n = 253) from one commercial beef farm in Rondônia - Brazil, were used in this study. All cows were maintained on Brachiaria brizantha pasture and given mineralized-salt and free access to water. Cows were treated according the experimental design depicted in Figure 1. The TAI protocols were initiated between 30 and 60 d postpartum. Cows were randomly divided into two groups according to the protocol of TAI. The 8d group (n = 124; 60 primiparous and 64 multiparous cows), received 2 mg of estradiol benzoate (Sincrodiol®, Ouro Fino, Cravinhos, Brazil) i.m., and an intravaginal progesterone device (Sincrogest®, Ouro Fino, Cravinhos, Brazil) to synchronize time of follicular wave emergence on Day 0. The progesterone device was removed and cows were given 500 μg of cloprostenol sodium (Sincrocio®, Ouro Fino, Cravinhos, Brazil) i.m., 1 mg of estradiol cipionate (SincroCP®, Ouro Fino, Cravinhos, Brazil) i.m., and 300 IU of eCG (Sincro eCG®, Ouro Fino, Cravinhos, Brazil) i.m. on Day 8. Cows in the 9d group (n = 129; 60 primiparous and 69 multiparous) were treated using the same protocol on Day 0 as cows in the 8d group. Cows of this group, however, were injected with 500 μg of cloprostenol sodium i.m. on Day 7 and the progesterone insert was removed on Day 9 along with the injection of 1 mg of estradiol cipionate i.m., and 300 IU of eCG i.m.. On the morning of Day 10 in the 8d group, and of Day 11 in the 9d group (08:00 am), the diameter of the pre-ovulatory follicle (POF) was assessed by ultrasonography (SIUI CTS-900, linear probe with 5 MHZ, Guangdong, China) and cows from both groups were subsequently divided based on the size of the POF in the manner previously described by Pfeifer et al. (2015). Cows from both groups were inseminated once at one of the following time points, according to the
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diameter of the POF, as described previously (Pfeifer et al., 2015): Block 0 (POF ≥ 15 mm, TAI 0 h after conventional TAI), Block 1 (POF = 13 to 14.9 mm, TAI 6 h after conventional TAI), Block 2 (POF = 10.1 to 12.9 mm, 24 h after conventional TAI) and Block 3 (POF ≤ 10 mm, TAI 30 h after conventional TAI). Further ultrasonic examinations were performed 30 d post-TAI to assess pregnancy status. Visualization of the embryonic vesicle and detection of the embryo were the positive criteria for determining pregnancy. 2.4. Statistical analysis All statistical analyses were performed using the SAS 9.0 software (SAS Institute Inc., Cary, NC, USA). Diameter of the ovulatory follicle was analyzed by the general linear model (GLM) procedure and Tukey’s post-hoc test was used to determine difference among protocols. The body weight, age, BCS, and days postpartum did not have a significant effect when included in the model and were, therefore, excluded from the final statistical model. Protocol and parity were included in the final model in all analyses. Pregnancy per AI was evaluated by Chi-square test. Moreover, another Chisquare test was performed to evaluate the effects of protocol (8d compared with 9d) within each animal category and within each block. The P/AI was calculated as the proportion of cows pregnant 30 days after TAI divided by the number of cows that were inseminated. Differences among groups were considered significant based on a P-value of less than 0.05. The P-values between 0.05 and 0.10 were considered to be indicative of a trend. 3. Results There were no differences on P/AI between 8d (49.2%, 61 of 124) and 9d groups (45%, 58/129; P = 0.5). The ovarian responses indicated that cows from the 9d groups tended to have a larger POF (13 ± 0.2 mm) than cows from the 8d groups (12.3 ± 0.2 mm; P = 0.07). Moreover, multiparous cows had a larger POF (13.7 ± 0.2 mm) than
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primiparous cows (11.5 ± 0.2 mm; P < 0.001) considering those assigned to both 8d and 9d protocols. When primiparous and multiparous cows were analyzed separately, the protocol did not affect P/AI in primiparous cows (P = 0.45), however, multiparous cows from the 8d group tended to have a greater P/AI than cows from the 9d group (P = 0.08; Table 1). Data for the P/AI for each block (0-3) according to the protocol are included in Table 2. No difference was detected in P/AI between protocols, except in block 2, in which the 8d group had a greater P/AI than the 9d group (P = 0.02). 4. Discussion In the present study no difference was observed on P/AI for cows subjected to AIB using either 8d or 9d estradiol-progesterone based protocols. The P/AI observed in this study (~ 50%), in which all cows were inseminated using an AIB technique for timing the insemination based on ovulatory follicle size, was consistent with what others observed when using a conventional TAI procedure in early postpartum Nelore cows (Baruselli et al., 2004; Meneghetti et al., 2009; Sa Filho et al., 2010). Based on a previous study, however, it was observed that postponing the time of TAI according to the diameter of the POF could improve pregnancy rates, especially in cows with smaller follicles (Pfeifer et al., 2015). It is important to note that the previous study (Pfeifer et al., 2015) was performed using only multiparous cows while using the 8d protocol. Considering this, the P/AI detected in multiparous cows when using the 8d protocol in the present study (64%) was consistent with results of the previous study. These findings suggest, therefore, that AIB may be more effective for multiparous cows by using the 8d estradiol-progesterone based protocol, however, more studies are needed to confirm this hypothesis. This finding was expected, however, because the AIB technique was developed and validated to be used in multiparous cows (Pfeifer et al., 2015).
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The 8d and 9d protocols used in the present study are the most used protocols for timed AI cows in South American beef operations. The use of these protocols is more efficient in postpartum cows located in tropical climates than those based on synchronization of time of follicular wave emergence with GnRH (Baruselli et al., 2002), resulting in satisfactory results in both estrous acyclic and cyclic beef cows (Rhodes et al., 2002). The P/AI observed in the present study was consistent with previous results where there were 54% (Baruselli et al., 2004) and ~ 55% pregnancy rates (Meneghetti et al., 2009), in lactating Nelore cows treated using the 8d and 9d estradiol-progesterone based protocols, respectively. The overall P/AI using the AIB technique while using the 8d or 9d estradiol-progesterone based protocol was not different, suggesting it can be efficiently used with both protocol regimens. It was, however, observed that cows in the present study of the 9d protocol group tended to develop a larger POF than cows in the 8d protocol group. Similarly, multiparous cows also developed a larger POF than primiparous cows. The relationship between the diameter of the dominant follicle and the interval to ovulation is well characterized in progesterone based protocols (Bo et al., 2003; Martinez et al., 2000; Martinez et al., 2005). Use of the AIB procedure, therefore, could be applied to different protocols and animal categories (heifers and primiparous and multiparous cows) using the same regimens. In addition to the diameter of the POF, the time of ovulation depends on interactions among hormones (Fortune et al., 2009), follicle maturation (Austin et al., 1999; Burke et al., 2001), proestrus length (Bridges et al., 2010), and time of acquisition of ovulatory capacity (Gimenes et al., 2008; Martinez et al., 1999; Sartori et al., 2001). Further studies aiming to develop a more appropriate AIB protocol for different animal categories and FTAI protocols are, therefore, necessary to achieve more desirable pregnancy results. Although the TAI protocols used in the present study are very effective and wellestablished methods have been developed to induce synchronized times of follicular wave emergence among cows (Baruselli et al., 2004; Bo et al., 2002; Meneghetti et al.,
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2009), there is still great variability in the follicle size at the end of the protocol treatment period (Bo et al., 2003). A protocol which consists in 9 d of progesterone treatment, in which luteolysis was induced 2 d before insert removal may increase the length of proestrus and the interval from follicular wave emergence to ovulation. This may explain why the majority of cows (66.7%) from the 9d group were inseminated in Blocks 0 and 1, and only 46.8% of the cows from 8d group were inseminated at this time point. This should not, however, affect the results with use of the AIB technique, because cows with smaller follicles are inseminated later and, therefore, use of this treatment protocol also results in increased P/AI compared to the use of the conventional technique. The previous findings for time of insemination are in agreement with observations in the present study because no overall difference was observed between the 8d and 9d protocol. Overall, no difference in the P/AI was detected between primiparous and multiparous cows in the present study. Within the 8d group, however, multiparous cows had a greater P/AI in comparison to primiparous cows. One could speculate that the regimen of AIB used in the present experiment is not adequate for primiparous cows. To answer this question future experiments are necessary and more field studies are underway to further expand on the findings of the present study. Although, in some studies there were no differences in fertility between multiparous and primiparous Nelore postpartum cows (Marques et al., 2015; Meneghetti et al., 2009; Sa Filho et al., 2010), others have found that multiparous lactating beef cows are more likely to become pregnant with use of TAI programs (Ferraz Junior et al., 2016; Sa Filho et al., 2013). In the present study, 31% (37/120) of the primiparous cows were included in the Block 3 AI time period. In contrast, in multiparous cows, only 12% (16/133) of cows were included in this AI time block. This occurred probably due the smaller follicles observed in primiparous compared to multiparous cows.
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In conclusion, in the present study no difference was observed on P/AI for cows subjected to the AIB time block treatment protocol using either 8d or 9d estradiolprogesterone based treatments. Multiparous cows, inseminated when using the 8d protocol and the AIB technique tended to have a greater P/AI in the present study but more studies are necessary to confirm this finding, and to develop protocols for timing of AI that are specific for each parity and duration of progesterone treatment.
Acknowledgements The authors would like to thank the staff of Embrapa’s Research Farm (Porto Velho, RO, Brazil) and of Fazenda São José (Jaru, RO, Brazil) for their help and for allowing the use of animals and facilities for this study. This research was supported by Embrapa (MP1/PC3 Project; 01.13.06.001.03.00). NAC was supported by CAPES from the Ministry of Education, and, JPC, and PMAN were supported by Cnpq from the Ministry of Science, Technology and Innovation of Brazil.
References ASBIA, 2011. Associação Brasileira de Inseminacao Artificial (Brazillian Society of Artificial Insemination). Austin, E.J., Mihm, M., Ryan, M.P., Williams, D.H., Roche, J.F., 1999. Effect of duration of dominance of the ovulatory follicle on onset of estrus and fertility in heifers. J. Anim. Sci. 77, 2219-2226. Baruselli, P.S., Marques, M.O., Carvalho, N.A.T., Madureira, E.H., Campos Filho, E.P., 2002. Efeitos de diferentes protocolos de inseminação artificial em tempo fixo na eficiência reprodutiva de vacas de corte lactantes. Rev. Bras. Reprod. Anim. 26, 218-221. Baruselli, P.S., Reis, E.L., Marques, M.O., Nasser, L.F., Bo, G.A., 2004. The use of hormonal treatments to improve reproductive performance of anestrous beef cattle in tropical climates. Anim. Reprod. Sci. 82-83, 479-486. Bo, G.A., Adams, G.P., Pierson, R.A., Tríbulo, H., Caccia, M., Mapletoft, R.J., 1994. Follicular wave dynamics after estradiol-17 β treatment of heifers with or without a progestogen implant. Theriogenology 41, 1555-1569.
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Bo, G.A., Baruselli, P.S., Moreno, D., Cutaia, L., Caccia, M., Tribulo, R., Tribulo, H., Mapletoft, R.J., 2002. The control of follicular wave development for selfappointed embryo transfer programs in cattle. Theriogenology 57, 53-72. Bo, G.A., Baruselli, P.S., Martinez, M.F., 2003. Pattern and manipulation of follicular development in Bos indicus cattle. Anim. Reprod. Sci. 78, 307-326. Bo, G.A., de la Mata, J.J., Baruselli, P.S., Menchaca, A., 2016. Alternative programs for synchronizing and resynchronizing ovulation in beef cattle. Theriogenology 86, 388-396. Bridges, G.A., Mussard, M.L., Burke, C.R., Day, M.L., 2010. Influence of the length of proestrus on fertility and endocrine function in female cattle. Anim. Reprod. Sci. 117, 208-215. Burke, C.R., Mussard, M.L., Grum, D.E., Day, M.L., 2001. Effects of maturity of the potential ovulatory follicle on induction of oestrus and ovulation in cattle with oestradiol benzoate. Anim. Reprod. Sci. 66, 161-174. Day, M.L., 2015. State of the art of GnRH-based timed Ai in beef cattle. Anim. Reprod. 12, 473-478. Ferraz Junior, M.V., Pires, A.V., Biehl, M.V., Santos, M.H., Barroso, J.P., Goncalves, J.R., Sartori, R., Day, M.L., 2016. Comparison of two timed artificial insemination system schemes to synchronize estrus and ovulation in Nellore cattle. Theriogenology. Fortune, J.E., Willis, E.L., Bridges, P.J., Yang, C.S., 2009. The periovulatory period in cattle: progesterone, prostaglandins, oxytocin and ADAMTS proteases. Anim. Reprod. 6, 60-71. Gimenes, L.U., Sa Filho, M.F., Carvalho, N.A., Torres-Junior, J.R., Souza, A.H., Madureira, E.H., Trinca, L.A., Sartorelli, E.S., Barros, C.M., Carvalho, J.B., Mapletoft, R.J., Baruselli, P.S., 2008. Follicle deviation and ovulatory capacity in Bos indicus heifers. Theriogenology 69, 852-858. Marques, M.O., Morotti, F., da Silva, C.B., Junior, M.R., da Silva, R.C., Baruselli, P.S., Seneda, M.M., 2015. Influence of category--heifers, primiparous and multiparous lactating cows--in a large-scale resynchronization fixed-time artificial insemination program. J. Vet. Sci. 16, 367-371. Martinez, M.F., Adams, G.P., Bergfelt, D.R., Kastelic, J.P., Mapletoft, R.J., 1999. Effect of LH or GnRH on the dominant follicle of the first follicular wave in beef heifers. Anim. Reprod. Sci. 57, 23-33. Martinez, M.F., Adams, G.P., Kastelic, J.P., Bergfel, D.R., Mapletoft, R.J., 2000. Induction of follicular wave emergence for estrus synchronization and artificial insemination in heifers. Theriogenology 54, 757-769. Martinez, M.F., Kastelic, J.P., Bo, G.A., Caccia, M., Mapletoft, R.J., 2005. Effects of oestradiol and some of its esters on gonadotrophin release and ovarian follicular dynamics in CIDR-treated beef cattle. Anim. Reprod. Sci. 86, 37-52. Meneghetti, M., Sa Filho, O.G., Peres, R.F., Lamb, G.C., Vasconcelos, J.L., 2009. Fixedtime artificial insemination with estradiol and progesterone for Bos indicus cows I: basis for development of protocols. Theriogenology 72, 179-189. Pfeifer, L.F., Castro, N.A., Melo, V.T., Neves, P.M., Cestaro, J.P., Schneider, A., 2015. Timed artificial insemination in blocks: A new alternative to improve fertility in lactating beef cows. Anim. Reprod. Sci. 163, 89-96. Pursley, J.R., Mee, M.O., Wiltbank, M.C., 1995. Synchronization of ovulation in dairy cows using PGF2alpha and GnRH. Theriogenology 44, 915-923.Randel, R.D., 1990. Nutrition and Postpartum Rebreeding in Cattle. J. Anim. Sci. 68, 853-862. Rhodes, F.M., Burke, C.R., Clark, B.A., Day, M.L., Macmillan, K.L., 2002. Effect of treatment with progesterone and oestradiol benzoate on ovarian follicular turnover in postpartum anoestrous cows and cows which have resumed oestrous cycles. Anim. Reprod. Sci. 69, 139-150. Sa Filho, M.F., Crespilho, A.M., Santos, J.E., Perry, G.A., Baruselli, P.S., 2010. Ovarian follicle diameter at timed insemination and estrous response influence likelihood 10
of ovulation and pregnancy after estrous synchronization with progesterone or progestin-based protocols in suckled Bos indicus cows. Anim. Reprod. Sci. 120, 23-30. Sa Filho, M.F., Penteado, L., Reis, E.L., Reis, T.A., Galvao, K.N., Baruselli, P.S., 2013. Timed artificial insemination early in the breeding season improves the reproductive performance of suckled beef cows. Theriogenology 79, 625-632. Sartori, R., Fricke, P.M., Ferreira, J.C., Ginther, O.J., Wiltbank, M.C., 2001. Follicular deviation and acquisition of ovulatory capacity in bovine follicles. Biol. Reprod. 65, 1403-1409. Whittier, W.D., Currin, J.F., Schramm, H., Holland, S., Kasimanickam, R.K., 2013. Fertility in Angus cross beef cows following 5-day CO-Synch + CIDR or 7-day CO-Synch + CIDR estrus synchronization and timed artificial insemination. Theriogenology 80, 963-969.
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Fig. 1. Experimental design used for suckled cows treated with 8d or 9d EB-CIDR based TAI protocol EB, estradiol benzoate; eCG, equine chorionic gonadotropin; ECP, estradiol cypionate; PGF, prostaglandin; TAI, timed artificial insemination; US, ultrassound examinations
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Table 1: Pregnancy per AI of primiparous and multiparous cows inseminated in blocks treated with 8d and 9d CIDR-EB based protocols Protocol 8d 9d P-value1 Overall 49.2% (61/124) 45% (58/129) 0.5 Primiparous 33.3% (20/60) 40% (24/60) 0.45 Multiparous 64.1% (41/64) 49.3% (34/69) 0.08 1 Values of P that indicate the difference between protocols within each category
Table 2: Pregnancy per AI of cows treated with 8d and 9d CIDR-EB based protocols according to the blocks of insemination Protocol Block
8d
9d
P-value
0 (AI immediately)
61% (14/23)
50% (19/38)
0.4
1 (AI + 6 h)
60% (21/35)
61% (28/46)
0.9
2 (AI + 24 h)
64.5% (20/31)
33.3% (9/27)
0.02
3 (AI +30 h)
17.1% (6/35)
11.1% (2/18)
0.7
13
S0378-4320(16)30586-3 http://dx.doi.org/doi:10.1016/j.anireprosci.2017.03.025 ANIREP 5577
To appear in:
Animal Reproduction Science
Received date: Accepted date:
25-10-2016 31-3-2017
Please cite this article as: Pfeifer, L.F.M., Castro, N.A., Neves, P.M.A., Cestaro, J.P., Schneider, A., Comparison between two estradiol-progesterone based protocols for timed artificial insemination in blocks in lactating Nelore cows.Animal Reproduction Science http://dx.doi.org/10.1016/j.anireprosci.2017.03.025 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Comparison between two estradiol-progesterone based protocols for timed artificial insemination in blocks in lactating Nelore cows L.F.M. Pfeifera*, N.A. Castrob, P.M.A. Nevesc, J.P. Cestaroc, A. Schneiderb Embrapa, Brazilian Agricultural Research Corporation, Porto Velho, Rondônia, Brazil
a
b
Departamento de Nutrição, Universidade Federal de Pelotas, Pelotas, Rio Grande do
Sul, Brazil FIMCA, Faculdades Integradas Aparício Carvalho, Porto Velho, Rondônia, Brazil
c
*Corresponding Author: Luiz Francisco Machado Pfeifer; Embrapa Rondônia, BR 364 Km 5,5 - Zona Rural, Caixa postal: 127 CEP: 76815-800, Porto Velho - Rondônia – Brazil; Business phone: 01155 69 3901-2510; E-mail: [email protected]
1
ABSTRACT The aim of this study was to compare the use of artificial insemination in time blocks (Artificial Insemination Blocks, AIB) using an 8 and 9 d estradiol-progesterone based protocol. In this experiment, lactating Nelore cows (n = 253) were subjected to two estradiol-progesterone based TAI protocols. On the morning of Day 10 (8d group, n = 124) or Day 11 (9d group, n = 129), cows were examined by ultrasonography to evaluate the diameter of the preovulatory follicle and were inseminated once at one of the following time points, according to the diameter of the pre-ovulatory follicle (POF): Block 0 (POF ≥ 15 mm, TAI 0 h after conventional TAI), Block 1 (POF 13.0 to 14.9 mm, TAI 6 h later), Block 2 (POF 10.1 to 12.9 mm, TAI 24 h later), and Block 3 (POF ≤ 10.0 mm, TAI 30 h later). The pregnancy per AI (P/AI) did not differ between 8d and 9d groups (P > 0.05). Considering only multiparous cows, however, P/AI tended to be greater in the 8d (64.1%) than in the 9d group (49.3%; P = 0.08). Cows from the 9d group tended to have a larger POF than cows from the 8d group (P = 0.07). In conclusion, these results provide evidence that there is no difference between 8d or 9d protocols when using the AIB technique. Use of the 8d estradiol-progesterone based protocol, however, tended to increase pregnancy in multiparous cows.
Keywords: Cattle; Follicular diameter; Ovulation; Ultrasonography
1. Introduction Artificial insemination (AI) is one of the main techniques used worldwide to disseminate desirable genetics among beef and dairy herds (Bo et al., 2016) and the development of timed AI (TAI) protocols contributed to the widespread use of the AI technique. In Brazil, semen commercialization increased 300% between 2000 and 2011 due to use of TAI (ASBIA, 2011). Data collected from the 2015 breeding season suggest
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that the number of cattle involved in TAI programs was about 10,000,000 in Brazilian commercial herds (Bo et al., 2016). Since the first report of the Ovsynch (Pursley et al., 1995) and estradiolprogesterone based protocols (Bo et al., 1994), estrous synchronization programs have been modified to improve fertility response of inseminated cows. However, only small improvements in the fertility of cows occurred when TAI protocols were used (Bo et al., 2016; Day, 2015; Meneghetti et al., 2009; Sa Filho et al., 2010; Sa Filho et al., 2013; Whittier et al., 2013). The most used protocol for Nelore cows maintained in tropical areas consists of an intravaginal progesterone insert, estradiol benzoate (EB) to induce synchronous ovarian follicular wave emergence, prostaglandin F2a analogues (PGFs) to induce luteolysis, and an ovulatory stimulus using an ester of estradiol (benzoate or cypionate). This estradiol-based protocol and its modifications provided satisfactory fertility in commercial Brazilian herds, independent of breed (B. indicus, B. taurus, or crossbreds). The 8d and 9d estradiol-progesterone based protocols were described previously by Baruselli et al. (2004) and Meneghetti et al. (2009), respectively, and are recognized as the most used protocols for TAI in lactating Nelore cows in Brazil. Currently, such TAI protocols result in pregnancy rates per AI (P/AI) that range from 40% to 60%, therefore, new alternatives to increase fertility of cows submitted to TAI programs are necessary. Recently, a technique known as AI in time blocks (Artificial Insemination Blocks, AIB) was developed in attempts to increase fertility with use of estradiolprogesterone based protocols for TAI (Pfeifer et al., 2015). The use of AIB protocols resulted in a P/AI of 60% in lactating Nelore cows. Because the AIB technique was developed to be used in 8d estradiol-progesterone based protocol, there are questions if the same blocking system for time of AI would be effective with use of 9d protocols. Based on these considerations, the objective of the present study was to compare the P/AI for cows inseminated in time blocks based on size of the largest follicle using an 8d and 9d protocol.
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2. Materials and methods The Committee for Ethics in Animal Experimentation from the Embrapa approved all of the procedures performed in the experiments described in this manuscript (Protocol F02.2014). 2.1. Animal treatments Lactating beef cows (B. indicus, Nelore; n = 253) from one commercial beef farm in Rondônia - Brazil, were used in this study. All cows were maintained on Brachiaria brizantha pasture and given mineralized-salt and free access to water. Cows were treated according the experimental design depicted in Figure 1. The TAI protocols were initiated between 30 and 60 d postpartum. Cows were randomly divided into two groups according to the protocol of TAI. The 8d group (n = 124; 60 primiparous and 64 multiparous cows), received 2 mg of estradiol benzoate (Sincrodiol®, Ouro Fino, Cravinhos, Brazil) i.m., and an intravaginal progesterone device (Sincrogest®, Ouro Fino, Cravinhos, Brazil) to synchronize time of follicular wave emergence on Day 0. The progesterone device was removed and cows were given 500 μg of cloprostenol sodium (Sincrocio®, Ouro Fino, Cravinhos, Brazil) i.m., 1 mg of estradiol cipionate (SincroCP®, Ouro Fino, Cravinhos, Brazil) i.m., and 300 IU of eCG (Sincro eCG®, Ouro Fino, Cravinhos, Brazil) i.m. on Day 8. Cows in the 9d group (n = 129; 60 primiparous and 69 multiparous) were treated using the same protocol on Day 0 as cows in the 8d group. Cows of this group, however, were injected with 500 μg of cloprostenol sodium i.m. on Day 7 and the progesterone insert was removed on Day 9 along with the injection of 1 mg of estradiol cipionate i.m., and 300 IU of eCG i.m.. On the morning of Day 10 in the 8d group, and of Day 11 in the 9d group (08:00 am), the diameter of the pre-ovulatory follicle (POF) was assessed by ultrasonography (SIUI CTS-900, linear probe with 5 MHZ, Guangdong, China) and cows from both groups were subsequently divided based on the size of the POF in the manner previously described by Pfeifer et al. (2015). Cows from both groups were inseminated once at one of the following time points, according to the
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diameter of the POF, as described previously (Pfeifer et al., 2015): Block 0 (POF ≥ 15 mm, TAI 0 h after conventional TAI), Block 1 (POF = 13 to 14.9 mm, TAI 6 h after conventional TAI), Block 2 (POF = 10.1 to 12.9 mm, 24 h after conventional TAI) and Block 3 (POF ≤ 10 mm, TAI 30 h after conventional TAI). Further ultrasonic examinations were performed 30 d post-TAI to assess pregnancy status. Visualization of the embryonic vesicle and detection of the embryo were the positive criteria for determining pregnancy. 2.4. Statistical analysis All statistical analyses were performed using the SAS 9.0 software (SAS Institute Inc., Cary, NC, USA). Diameter of the ovulatory follicle was analyzed by the general linear model (GLM) procedure and Tukey’s post-hoc test was used to determine difference among protocols. The body weight, age, BCS, and days postpartum did not have a significant effect when included in the model and were, therefore, excluded from the final statistical model. Protocol and parity were included in the final model in all analyses. Pregnancy per AI was evaluated by Chi-square test. Moreover, another Chisquare test was performed to evaluate the effects of protocol (8d compared with 9d) within each animal category and within each block. The P/AI was calculated as the proportion of cows pregnant 30 days after TAI divided by the number of cows that were inseminated. Differences among groups were considered significant based on a P-value of less than 0.05. The P-values between 0.05 and 0.10 were considered to be indicative of a trend. 3. Results There were no differences on P/AI between 8d (49.2%, 61 of 124) and 9d groups (45%, 58/129; P = 0.5). The ovarian responses indicated that cows from the 9d groups tended to have a larger POF (13 ± 0.2 mm) than cows from the 8d groups (12.3 ± 0.2 mm; P = 0.07). Moreover, multiparous cows had a larger POF (13.7 ± 0.2 mm) than
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primiparous cows (11.5 ± 0.2 mm; P < 0.001) considering those assigned to both 8d and 9d protocols. When primiparous and multiparous cows were analyzed separately, the protocol did not affect P/AI in primiparous cows (P = 0.45), however, multiparous cows from the 8d group tended to have a greater P/AI than cows from the 9d group (P = 0.08; Table 1). Data for the P/AI for each block (0-3) according to the protocol are included in Table 2. No difference was detected in P/AI between protocols, except in block 2, in which the 8d group had a greater P/AI than the 9d group (P = 0.02). 4. Discussion In the present study no difference was observed on P/AI for cows subjected to AIB using either 8d or 9d estradiol-progesterone based protocols. The P/AI observed in this study (~ 50%), in which all cows were inseminated using an AIB technique for timing the insemination based on ovulatory follicle size, was consistent with what others observed when using a conventional TAI procedure in early postpartum Nelore cows (Baruselli et al., 2004; Meneghetti et al., 2009; Sa Filho et al., 2010). Based on a previous study, however, it was observed that postponing the time of TAI according to the diameter of the POF could improve pregnancy rates, especially in cows with smaller follicles (Pfeifer et al., 2015). It is important to note that the previous study (Pfeifer et al., 2015) was performed using only multiparous cows while using the 8d protocol. Considering this, the P/AI detected in multiparous cows when using the 8d protocol in the present study (64%) was consistent with results of the previous study. These findings suggest, therefore, that AIB may be more effective for multiparous cows by using the 8d estradiol-progesterone based protocol, however, more studies are needed to confirm this hypothesis. This finding was expected, however, because the AIB technique was developed and validated to be used in multiparous cows (Pfeifer et al., 2015).
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The 8d and 9d protocols used in the present study are the most used protocols for timed AI cows in South American beef operations. The use of these protocols is more efficient in postpartum cows located in tropical climates than those based on synchronization of time of follicular wave emergence with GnRH (Baruselli et al., 2002), resulting in satisfactory results in both estrous acyclic and cyclic beef cows (Rhodes et al., 2002). The P/AI observed in the present study was consistent with previous results where there were 54% (Baruselli et al., 2004) and ~ 55% pregnancy rates (Meneghetti et al., 2009), in lactating Nelore cows treated using the 8d and 9d estradiol-progesterone based protocols, respectively. The overall P/AI using the AIB technique while using the 8d or 9d estradiol-progesterone based protocol was not different, suggesting it can be efficiently used with both protocol regimens. It was, however, observed that cows in the present study of the 9d protocol group tended to develop a larger POF than cows in the 8d protocol group. Similarly, multiparous cows also developed a larger POF than primiparous cows. The relationship between the diameter of the dominant follicle and the interval to ovulation is well characterized in progesterone based protocols (Bo et al., 2003; Martinez et al., 2000; Martinez et al., 2005). Use of the AIB procedure, therefore, could be applied to different protocols and animal categories (heifers and primiparous and multiparous cows) using the same regimens. In addition to the diameter of the POF, the time of ovulation depends on interactions among hormones (Fortune et al., 2009), follicle maturation (Austin et al., 1999; Burke et al., 2001), proestrus length (Bridges et al., 2010), and time of acquisition of ovulatory capacity (Gimenes et al., 2008; Martinez et al., 1999; Sartori et al., 2001). Further studies aiming to develop a more appropriate AIB protocol for different animal categories and FTAI protocols are, therefore, necessary to achieve more desirable pregnancy results. Although the TAI protocols used in the present study are very effective and wellestablished methods have been developed to induce synchronized times of follicular wave emergence among cows (Baruselli et al., 2004; Bo et al., 2002; Meneghetti et al.,
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2009), there is still great variability in the follicle size at the end of the protocol treatment period (Bo et al., 2003). A protocol which consists in 9 d of progesterone treatment, in which luteolysis was induced 2 d before insert removal may increase the length of proestrus and the interval from follicular wave emergence to ovulation. This may explain why the majority of cows (66.7%) from the 9d group were inseminated in Blocks 0 and 1, and only 46.8% of the cows from 8d group were inseminated at this time point. This should not, however, affect the results with use of the AIB technique, because cows with smaller follicles are inseminated later and, therefore, use of this treatment protocol also results in increased P/AI compared to the use of the conventional technique. The previous findings for time of insemination are in agreement with observations in the present study because no overall difference was observed between the 8d and 9d protocol. Overall, no difference in the P/AI was detected between primiparous and multiparous cows in the present study. Within the 8d group, however, multiparous cows had a greater P/AI in comparison to primiparous cows. One could speculate that the regimen of AIB used in the present experiment is not adequate for primiparous cows. To answer this question future experiments are necessary and more field studies are underway to further expand on the findings of the present study. Although, in some studies there were no differences in fertility between multiparous and primiparous Nelore postpartum cows (Marques et al., 2015; Meneghetti et al., 2009; Sa Filho et al., 2010), others have found that multiparous lactating beef cows are more likely to become pregnant with use of TAI programs (Ferraz Junior et al., 2016; Sa Filho et al., 2013). In the present study, 31% (37/120) of the primiparous cows were included in the Block 3 AI time period. In contrast, in multiparous cows, only 12% (16/133) of cows were included in this AI time block. This occurred probably due the smaller follicles observed in primiparous compared to multiparous cows.
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In conclusion, in the present study no difference was observed on P/AI for cows subjected to the AIB time block treatment protocol using either 8d or 9d estradiolprogesterone based treatments. Multiparous cows, inseminated when using the 8d protocol and the AIB technique tended to have a greater P/AI in the present study but more studies are necessary to confirm this finding, and to develop protocols for timing of AI that are specific for each parity and duration of progesterone treatment.
Acknowledgements The authors would like to thank the staff of Embrapa’s Research Farm (Porto Velho, RO, Brazil) and of Fazenda São José (Jaru, RO, Brazil) for their help and for allowing the use of animals and facilities for this study. This research was supported by Embrapa (MP1/PC3 Project; 01.13.06.001.03.00). NAC was supported by CAPES from the Ministry of Education, and, JPC, and PMAN were supported by Cnpq from the Ministry of Science, Technology and Innovation of Brazil.
References ASBIA, 2011. Associação Brasileira de Inseminacao Artificial (Brazillian Society of Artificial Insemination). Austin, E.J., Mihm, M., Ryan, M.P., Williams, D.H., Roche, J.F., 1999. Effect of duration of dominance of the ovulatory follicle on onset of estrus and fertility in heifers. J. Anim. Sci. 77, 2219-2226. Baruselli, P.S., Marques, M.O., Carvalho, N.A.T., Madureira, E.H., Campos Filho, E.P., 2002. Efeitos de diferentes protocolos de inseminação artificial em tempo fixo na eficiência reprodutiva de vacas de corte lactantes. Rev. Bras. Reprod. Anim. 26, 218-221. Baruselli, P.S., Reis, E.L., Marques, M.O., Nasser, L.F., Bo, G.A., 2004. The use of hormonal treatments to improve reproductive performance of anestrous beef cattle in tropical climates. Anim. Reprod. Sci. 82-83, 479-486. Bo, G.A., Adams, G.P., Pierson, R.A., Tríbulo, H., Caccia, M., Mapletoft, R.J., 1994. Follicular wave dynamics after estradiol-17 β treatment of heifers with or without a progestogen implant. Theriogenology 41, 1555-1569.
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Bo, G.A., Baruselli, P.S., Moreno, D., Cutaia, L., Caccia, M., Tribulo, R., Tribulo, H., Mapletoft, R.J., 2002. The control of follicular wave development for selfappointed embryo transfer programs in cattle. Theriogenology 57, 53-72. Bo, G.A., Baruselli, P.S., Martinez, M.F., 2003. Pattern and manipulation of follicular development in Bos indicus cattle. Anim. Reprod. Sci. 78, 307-326. Bo, G.A., de la Mata, J.J., Baruselli, P.S., Menchaca, A., 2016. Alternative programs for synchronizing and resynchronizing ovulation in beef cattle. Theriogenology 86, 388-396. Bridges, G.A., Mussard, M.L., Burke, C.R., Day, M.L., 2010. Influence of the length of proestrus on fertility and endocrine function in female cattle. Anim. Reprod. Sci. 117, 208-215. Burke, C.R., Mussard, M.L., Grum, D.E., Day, M.L., 2001. Effects of maturity of the potential ovulatory follicle on induction of oestrus and ovulation in cattle with oestradiol benzoate. Anim. Reprod. Sci. 66, 161-174. Day, M.L., 2015. State of the art of GnRH-based timed Ai in beef cattle. Anim. Reprod. 12, 473-478. Ferraz Junior, M.V., Pires, A.V., Biehl, M.V., Santos, M.H., Barroso, J.P., Goncalves, J.R., Sartori, R., Day, M.L., 2016. Comparison of two timed artificial insemination system schemes to synchronize estrus and ovulation in Nellore cattle. Theriogenology. Fortune, J.E., Willis, E.L., Bridges, P.J., Yang, C.S., 2009. The periovulatory period in cattle: progesterone, prostaglandins, oxytocin and ADAMTS proteases. Anim. Reprod. 6, 60-71. Gimenes, L.U., Sa Filho, M.F., Carvalho, N.A., Torres-Junior, J.R., Souza, A.H., Madureira, E.H., Trinca, L.A., Sartorelli, E.S., Barros, C.M., Carvalho, J.B., Mapletoft, R.J., Baruselli, P.S., 2008. Follicle deviation and ovulatory capacity in Bos indicus heifers. Theriogenology 69, 852-858. Marques, M.O., Morotti, F., da Silva, C.B., Junior, M.R., da Silva, R.C., Baruselli, P.S., Seneda, M.M., 2015. Influence of category--heifers, primiparous and multiparous lactating cows--in a large-scale resynchronization fixed-time artificial insemination program. J. Vet. Sci. 16, 367-371. Martinez, M.F., Adams, G.P., Bergfelt, D.R., Kastelic, J.P., Mapletoft, R.J., 1999. Effect of LH or GnRH on the dominant follicle of the first follicular wave in beef heifers. Anim. Reprod. Sci. 57, 23-33. Martinez, M.F., Adams, G.P., Kastelic, J.P., Bergfel, D.R., Mapletoft, R.J., 2000. Induction of follicular wave emergence for estrus synchronization and artificial insemination in heifers. Theriogenology 54, 757-769. Martinez, M.F., Kastelic, J.P., Bo, G.A., Caccia, M., Mapletoft, R.J., 2005. Effects of oestradiol and some of its esters on gonadotrophin release and ovarian follicular dynamics in CIDR-treated beef cattle. Anim. Reprod. Sci. 86, 37-52. Meneghetti, M., Sa Filho, O.G., Peres, R.F., Lamb, G.C., Vasconcelos, J.L., 2009. Fixedtime artificial insemination with estradiol and progesterone for Bos indicus cows I: basis for development of protocols. Theriogenology 72, 179-189. Pfeifer, L.F., Castro, N.A., Melo, V.T., Neves, P.M., Cestaro, J.P., Schneider, A., 2015. Timed artificial insemination in blocks: A new alternative to improve fertility in lactating beef cows. Anim. Reprod. Sci. 163, 89-96. Pursley, J.R., Mee, M.O., Wiltbank, M.C., 1995. Synchronization of ovulation in dairy cows using PGF2alpha and GnRH. Theriogenology 44, 915-923.Randel, R.D., 1990. Nutrition and Postpartum Rebreeding in Cattle. J. Anim. Sci. 68, 853-862. Rhodes, F.M., Burke, C.R., Clark, B.A., Day, M.L., Macmillan, K.L., 2002. Effect of treatment with progesterone and oestradiol benzoate on ovarian follicular turnover in postpartum anoestrous cows and cows which have resumed oestrous cycles. Anim. Reprod. Sci. 69, 139-150. Sa Filho, M.F., Crespilho, A.M., Santos, J.E., Perry, G.A., Baruselli, P.S., 2010. Ovarian follicle diameter at timed insemination and estrous response influence likelihood 10
of ovulation and pregnancy after estrous synchronization with progesterone or progestin-based protocols in suckled Bos indicus cows. Anim. Reprod. Sci. 120, 23-30. Sa Filho, M.F., Penteado, L., Reis, E.L., Reis, T.A., Galvao, K.N., Baruselli, P.S., 2013. Timed artificial insemination early in the breeding season improves the reproductive performance of suckled beef cows. Theriogenology 79, 625-632. Sartori, R., Fricke, P.M., Ferreira, J.C., Ginther, O.J., Wiltbank, M.C., 2001. Follicular deviation and acquisition of ovulatory capacity in bovine follicles. Biol. Reprod. 65, 1403-1409. Whittier, W.D., Currin, J.F., Schramm, H., Holland, S., Kasimanickam, R.K., 2013. Fertility in Angus cross beef cows following 5-day CO-Synch + CIDR or 7-day CO-Synch + CIDR estrus synchronization and timed artificial insemination. Theriogenology 80, 963-969.
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Fig. 1. Experimental design used for suckled cows treated with 8d or 9d EB-CIDR based TAI protocol EB, estradiol benzoate; eCG, equine chorionic gonadotropin; ECP, estradiol cypionate; PGF, prostaglandin; TAI, timed artificial insemination; US, ultrassound examinations
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Table 1: Pregnancy per AI of primiparous and multiparous cows inseminated in blocks treated with 8d and 9d CIDR-EB based protocols Protocol 8d 9d P-value1 Overall 49.2% (61/124) 45% (58/129) 0.5 Primiparous 33.3% (20/60) 40% (24/60) 0.45 Multiparous 64.1% (41/64) 49.3% (34/69) 0.08 1 Values of P that indicate the difference between protocols within each category
Table 2: Pregnancy per AI of cows treated with 8d and 9d CIDR-EB based protocols according to the blocks of insemination Protocol Block
8d
9d
P-value
0 (AI immediately)
61% (14/23)
50% (19/38)
0.4
1 (AI + 6 h)
60% (21/35)
61% (28/46)
0.9
2 (AI + 24 h)
64.5% (20/31)
33.3% (9/27)
0.02
3 (AI +30 h)
17.1% (6/35)
11.1% (2/18)
0.7
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