Pregnancy rates of crossbred dairy cattle synchronized using GnRH and one injection of PGF2alpha versus two injections of PGF2alpha prior to insemination

F O K Mgongo, P Mujuni and A Kitambi

Sokoine University of Agriculture, Faculty of Veterinary Medicine, Department of Surgery and Theriogenology, P. O. Box 3020, Morogoro, Tanzania, Tanzania
fmgongo@suanet.ac.tz

Abstract

Prostaglandin (PG) F₂alpha  and Gonadotrophin releasing hormone (GnRH) are used to improve oestrus detection in artificial insemination (AI) programs. However, pregnancy rates after GnRH – PGF₂alpha administration are poor in crossbred dairy cattle partly due to asynchronous ovarian events. This study tested the theory that two injections of PGF₂alpha may increase pregnancy rates. Data on cows inseminated at spontaneous oestrus formed control group 1 (n = 1120) and those synchronized with prostaglandin PGF₂alpha and inseminated twice upon detection of oestrus formed group 2 (n = 166). Animals in group 3 (n = 47) and 4 (n = 47) received GnRH on Day 1 and PGF₂alpha on Day 7 and were inseminated twice upon detection of oestrus. Animals in group 3 received additional PGF₂alpha on Day 8. Starting on Day 8 oestrus behavioural signs were graded and progesterone as well as vagina mucus electrical resistance determined in all animals of group 3 and 4.

Pregnancy rates determined 60 to 70 days after AI were 58.8% in group 1, 60.2% in group 2, 69.8% in group 3 and 63.6% in group 4. It is concluded that administration of PGF₂alpha prior to AI improved pregnancy rates.

Keywords: crossbred cattle, double insemination, GnRH, prostaglandin

Introduction

Cost management of artificial insemination (AI) programs include reducing costs emanating from failures to become pregnant after AI and costs incurred during detecting of oestrus (Thatcher et al 2002; Cairoli et al 2006). One goal with any program is to attain maximum pregnancy rates at the lowest possible cost (Holm et al 2008). Double insemination protocols are therefore rarely used due to pregnancy rates being consistently similar to those after single inseminations (Wahome et al 1985; Mgongo 1988 and Stevenson et al 1990) or lower (Young and Henderson 1981). By and large double inseminations protocols undertaken in routine AI programs raise costs and may jeopardize chances of first inseminations (Mgongo1988; Prinzen et al 1991 and Dransfield et al 1998).

However, there are numerous cases in the field that demand double inseminations. Essentially these are cases due to heat stress, asynchronous ovarian events exhibiting incomplete or delayed luteolysis, weak or delayed oestrus or may simply be a part of a protocol of synchronization of oestrus. The rate of occurrence of such cases requiring double insemination is very high in the tropics, ranging between 4 and 30% (Dobson and Kamonpatana 1986 and Mgongo 1988). One of the most significant differences between cows bred once at spontaneous oestrus and cows requiring double insemination are the rates at which progesterone levels decline (Mgongo 1988). Researchers have claimed that double AI may benefit difficult cases particularly those associated with poor detection of oestrus (Thatcher et al 2002; Holm et al 2008).

Strategies taken to increase effectiveness and value of scheduled double inseminations include use of Gonadotrophin releasing hormone (GnRH) prior or after insemination. GnRH controls follicular development by inducing ovulation or luteinization (Lemaster et al 2001 and Thatcher et al 2002). However, the ability of GnRH to induce ovulation or luteinization is influenced by the dose and stage of follicular development at time of injection (DeJarnette et al 2001a, 2001b). Eight to 10 percent of cows treated with GnRH fail to respond in the designed manner by either exhibiting spontaneous oestrus prematurely or exhibiting asynchronous ovarian events (Taponen et al 2002). Results obtained after use of GnRH - PGF₂alpha protocols in Bos taurus cross Bos indicus cows show that premature oestrus and asynchronous ovarian events with weak or no oestrous signs are very high in tropics and therefore GnRH - PGF₂alpha protocols in crossbred cattle in tropical environments require refinement.

Animals not responding to GnRH - PGF₂alpha protocols by overt signs of oestrus are most likely responding to asynchronous ovarian events, which in the tropics is commonly associated with incomplete or delayed luteolysis (Mgongo et al 1990 and DeJarnette et al 2001a). In such cases, a second injection of PGF₂alpha within the anticipated period of occurrence of oestrus may augment conception rates by bringing luteolysis to completion (Kasimanickam et al 2009). Treatment with two prostaglandin injections at an 8 hour interval improves luteolysis than single injections (Archbald et al 1993 and Repasi et al 2005). The second insemination may compromise events between overt - oestrus - insemination and ovulation and thereby increase pregnancy rates.

The aim of this study was to test the hypothesis that a second injection of PGF₂alpha given within the same oestrus to complete luteolysis may increase pregnancy rates in dairy cattle of the tropics. In addition this study aimed at providing more information concerning efficacy of GnRH - PGF₂alpha combinations for induction of oestrus in crossbred cattle (i.e. Bos taurus males crossed to the small Tanzania short horn zebu females).

Materials and methods 

Study area

The study was conducted within the practice area for the Sokoine University of Agriculture veterinary clinic at Morogoro, Tanzania. Morogoro receives over 800 mm of rain per year. The rainfall pattern is bimodal with a short and a long rains season. Long rains occur between February and May while short rains occur in October and November. The rest of the year consists of the short dry season in January and the long dry season in June and September. Further information on the area is as given by Mujuni et al (1993).

Animals

Animals studied were sexually mature (over 4 years) Friesian and Ayrshire crosses with indigenous Tanzania short horn zebu. Cows were grazed in the mornings and evenings when temperatures were low and supplemented with concentrates containing maize, cotton seed cake, minerals and vitamins at milking time to meet standard nutrient requirements for dairy cattle. Morning milking was done between 06.00 and 08.00 hour and afternoon milking between 16.00 and 18.00 hour. All animals received regular veterinary care, which included vaccinations against common diseases.

Data on spontaneous oestrus (group 1)

Retrospective data covering a 20-year period were collected from farm records. Complete records for 1120 single inseminations were available for analysis. In conjunction with routine veterinary clinical practice animals in oestrus were reported to the clinic and animals were inseminated once upon detection of spontaneous oestrus and according to the a.m. / p.m. rule.

Data on induced oestrus (group 2)

Retrospective data covering a five-year period were collected from farm records. Complete records for 166 successful synchronizations with either 500µg cloprostenol (a synthetic analogue of PGF₂alpha; Estrumate^®, Imperial Chemical Industries, Cheshire, England) or 5ml Illiren^® (a synthetic analogue of PGF₂alpha_, Hoechst Rousset Vet, Frankfurt, Germany) were made available for analysis. In conjunction with routine veterinary herd health, animals diagnosed not pregnant were selected and treated. All inseminations were done twice upon detection of oestrus.

Data on animals injected with GnRH - PGF₂alpha (group 3 and 4)

At the beginning of the experiment, the cyclic status of each animal was determined through rectal examination. Ninety four cows with healthy uterine environment, that is with no history of dystocia, retained foetal membranes or vaginal abnormal discharge and having a fully grown corpus luteum detected at rectal palpation and confirmed retrospectively by progesterone determination, were selected (day of selection = Day 1). Oestruses were synchronized with an intramuscular (im) injection of 20 μg buserelin (an analogue of GnRH; Receptal^®, Hoechst Rousset Vet, Frankfurt, Germany) given on Day 1 followed later by an intramuscular injection of 500μg cloprostenol on Day 7. Cows were divided randomly into group 3 (n = 47) and 4 (n = 47). Group 3 animals received an additional im injection of 500μg cloprostenol (PGF₂alpha) on Day 8. Thirty six hours after the first treatment with PGF₂alfa the animals were examined daily in mornings and evenings for oestrous signs together with the farm workers. AI was undertaken upon detection of oestrus. Oestrus behavioural signs on day of insemination were subjectively graded and referred to as level 1 = when signs were absent, level 2 = when signs were questionable and level 3 = when signs were clear and animals stood to be mounted. In addition, vagina mucus electrical resistance was determined at the time of insemination by using an ohmmeter (Brunstmessgerate^®, Hauptner Co.; Solingen, Germany) and foremilk samples were taken prior to injection of drugs and at insemination. Progesterone was determined by use of a cowside enzyme immunoassay test kit (Target^®, A. Albrecht GmbH, Aulendorf, Germany) not later than 2 hours after collection of milk. Concentration of progesterone was classified as low, intermediate or high by comparison with standard colours provided by the kit.

Transrectal ultrasound examinations were done by a single operator using a realtime, B-mode scanner equipped with a 5 MHz linear array transducer (SonoAce^®, Kretz Technik AG, Tiefenbach 20, Austria) to score for follicles, corpus lutea and pregnancy. Pregnancy diagnosis was done 26 days after insemination and was repeated at 60 – 70 days following AI.

Statistical analysis

Binomial data were analyzed by chi-square (2 x 2 tables) tests using Statistical Analysis Systems (SAS  1999. The general linear model procedure (analysis of variance, t-test) was employed to compare the means and find out effects of month and season on the intervals between injection of prostaglandin and artificial inseminations and pregnancy. The results are expressed as means or percentages (± standard error of mean; SEM). The differences were considered significant at P ≤ 0.05.

Results 

A total of 1120 cows coming to oestrus spontaneously were inseminated between 1985 and 2005. This is the control group. Analysis of data according to season gives 482/1120 (43.0%) inseminations for the long rain season, 141 (12.6%) for the short dry, 315 (28.1%) for the long dry and 182 (16.3%) for the short rain season; with the difference between seasons being significant (P<0.05; n=1120).

Out of 1120 cows 658 (58.8 %) cows were confirmed pregnant. The number of cows submitted for insemination that became pregnant within season was 117 of 141 (82.9%) for the short dry season, 321 of 482 (66.6%) for the long rain, 146 out of 315 (46.3%) for the long dry and 74 of 182 (40.6%) for the short rain season.

A total of 166 induced oestrus cows in group 2 were injected with prostaglandin and visually observed for oestrus. The proportion of cows that exhibited overt signs of oestrus within the first 7 days of cloprostenol (PGF₂alpha) administration was 134 (80.7%). The median day at which oestrus was observed was Day 3.5 (84 hours) with a range of one to ten days. Out of 166 cows 100 (60.2%) were diagnosed pregnant.

Out of 47 cows in group 3 that had received GnRH- PGF₂alpha-PGF₂alpha 43 (91.5%) exhibited overt signs of oestrus with 32 (68.1%) exhibiting very clear grade 3 oestrus behavioural signs. Only four cows in this group did not exhibit oestrous signs at all and were not inseminated. Out of 43 cows exhibiting behavioral signs of oestrus in group 3, 30 (69.8%) became pregnant and comprised of twenty four cows with grade 3 signs and 6 with grade 2 (questionable to intermediate) oestrous signs. Vagina mucus resistance in all 43 cows were bellow 30; with ranges between 25 and 30.

Out of 47 cows in group 4 that received GnRH- PGF_2alpha- (second PGF_2alpha- omitted), a total of 44 (93.6%) exhibited overt signs of oestrus with 26 (55.3%) exhibiting clear grade 3 signs and 18 (38.3%) exhibiting grade 2 (questionable to intermediate) signs of oestrus. Out of the 44 cows exhibiting very clear grade 3 oestrous behavioural signs in group 4, 28 (63.6%) became pregnant. The 28 pregnant in group 4 comprised of 26 cows with grade 3 oestrous signs and 8 with grade 2 (questionable to intermediate) oestrous signs. Further results are given in Text Table 1.

Further analysis of data to see if month and season of treatment had any significance to oestrous events in group 1 to 4 shows that month and season had some effect (P ≤ 0. 05;  n = 1120) on occurrence of oestrus in group one. In animals receiving cloprostenol (PGF_2alpha), that is, group 2 to 4, month of treatment had no effect on the interval between treatment and time of insemination (P ≥ 0.23; n = 94). Season had also no effect on the interval between cloprostenol (PGF₂alpha) and insemination in all the groups.

Discussion 

Using hormonal treatment to induce or to synchronize oestrus is often followed by a double insemination. In this study double inseminations were conducted after one injection of prostaglandin in group 2, after GnRH - PGF₂alpha _- PGF₂alpha injections in group 3 and GnRH - PGF₂alpha injections in group 4 and were compared to single inseminations undertaken during spontaneous oestrus. Distribution of pregnancy rates after synchronization in group 2 followed the same trend as that for spontaneous oestrus (group 1). Pregnancy rates were highest in animals with highest score of signs of oestrus. However, overall pregnancy rates were better than those after spontaneous oestrus. This can be explained by the timing of inseminations. Pregnancy rates were at 69.8% following double insemination in group 3. This is much higher than pregnancy rates in-group 4, which was at 63.6% after double inseminations. In view of the fact that synchronization was done during the long dry period, the period with minimum chance of pregnancy, the rate of 69.8% is significantly high. Usually double inseminations may have negative effects on conception by influencing uterine contractility through excessive handling of the uterus (Prinzen et al 1991). PGF₂alpha increases uterine contractility, compels completion of luteolysis, and through such actions synchronizes ovarian events, and increases pregnancy rates. This may explain benefits of the second dose of PGF₂alpha to double insemination in group 3. Usually complete luteolysis lead to higher conception rates than for partial luteolysis. Mgongo (1988) while working with double inseminations showed that decline in progesterone of more than 4ng per ml between Day 1 and Day 2 of contemporaneous oestrus increased conception rates. In this study progesterone values remained unchanged during the two days. Vaginal mucus resistance did not vary between Day 1 and Day 2. All in all strong to medium oestrus signs supported by low progesterone levels resulted in higher conception rates than weak heat signs. The boost in the exhibition of such signs in this study could be explained through use of an additional injection of PGF_2alpha and a second insemination.

 Results of the retrospective study (group 1) provide fundamental information on oestrus patterns in the tropics. Results show clearly that highest number of inseminations was done during the long rain and highest pregnancy rates occurred during the same season. Statistically, month of carrying out AI had no effect on success of AI and this is partly explained by the fact that once oestrus occurs spontaneously chances of conception are high. However, extrapolated from the rain pattern in Morogoro, the long rain period produced good pastures and consequently had had high occurrence of oestrus. These results conform to reports in the literature on influence of the tropical environment on occurrence of oestrus. The most important aspect of the environment is that there is a time lag between exposure and occurrence of effects. All in all occurrence of oestrus depends on prior and continued exposure to conductive environments, particularly to availability of pasture.

Analysis of pregnancy rates according to season gives the short dry (82.9%) and long rain (66.6 %) periods the highest scores. The long dry period with 46.30% and the short rain season with 40.6% were lower than the average (58.8%). Pregnancy rates in Morogoro are such that months with lots of rain and indirectly good pasture obtain highest pregnancy rates.

Conclusion 

• Although this study suffers from low numbers of animals per treatment group, it still indicates that, in tropical environments, oestrus synchronization and double insemination protocols may benefit from a second injection of PGF₂alpha. It is recommended however that experiments to involve a large population of cows be done to increase accuracy of this statement.

Acknowledgements 

I wish to acknowledge assistance provided by Prof. Dr. Rudolph Stolla of the Faculty of Veterinary Medicine, Munich, Germany, Hoechst Rousset Vet., Frankfurt, Germany and Germany Academic Exchange programme (DAAD) for supply of equipment and drugs.

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Received 19 May 2009; Accepted 12 June 2009; Published 5 August 2009

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