Medycyna Wet. 2008, 64 (2) 164
Praca oryginalna Original paper
The failure to detect cows in estrus accurately is a major factor limiting the reproductive performance of lactating dairy cattle and is the main reason for in-creasing the calving interval in many dairy herds (11). It has been estimated that the mean reduction in net revenue from a 1-d increase in adjusted calving inter-val is $ 4.7 (Canadian) per cow (10). Various oestrus synchronization programs allowing timed artificial in-semination (TAI) have been developed for cows but most require several days to sort cows and administer the treatment, and it is difficult to reduce the interval between the start of treatment and AI to less than 9 d (6). Moreover, despite progress in synchronizing oestrus, the reproductive performance of dairy cows has not substantially improved. Development of short synchronization methods that would result in accept-able pregnancy rates as a result of TAI would provide breeders with certain advantages. This would rid the breeders of the burden of determining the oestrus, all
cows treated following the voluntary waiting period would be inseminated, and early completion of treat-ment would save breeders time.
However, there are only a limited number of studies on the short synchronization programs for TAI, in which cows that had a determinable corpus luteum (CL) were used (5-7). According to our knowledge, there is only one study about the effects of short syn-chronization methods on random or known different stages of oestrus cycles in lactating cows (2). Further-more, there is no study in which gonadotropin rele-asing hormone (GnRH) or human chorionic gonado-tropin (hCG) is compared for the synchronization of ovulation in the short oestrus synchronization pro-grams. The simultaneous administration of hCG and estradiol benzoate 12 h after prostaglandin treatment in dairy cows with mature CL has been reported to allow TAI 48 h after starting treatment (6). In a recent study it has been shown that considerably
synchroni-Short oestrus synchronization with PGF
2a
, estradiol
and GnRH or hCG in lactating dairy cows
ÜMUT CIRIT, MUZAFFER TAÞ*, SULEYMAN BACINOGLU, OZEN BANU ÖZDAÞ, KEMAL AK, ÝRFAN KAMURAN ÝLERI
Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, Istanbul University, 34320 Avcilar, Istanbul, Turkey
*Department of Obstetrics and Reproduction, Faculty of Veterinary Medicine, Dicle University, 21280 Diyarbakir, Turkey Cirit Ü., Taþ M., Bacinoglu S., Özdaþ O. B., Ak K., Ýleri Ý. K.
Short oestrus synchronization with PGF2a, estradiol and GnRH or hCG in lactating dairy cows Summary
The objectives of this study were to develop short oestrus synchronization methods allowing timed artificial insemination (TAI) in lactating dairy cows and to compare the synchronizing effects of the use of human chorionic gonadotropin (hCG), instead of a gonadotropin releasing hormone (GnRH), on ovulation and pregnancy rates. An additional goal was to determine the effects of the presence or absence of an active corpus luteum (CL) on the treatment efficacy at the beginning of treatment. Sixty-three cows at random stages of the oestrus cycle on the 50th-95th postpartum day were randomly distributed into two groups. Cows of GnRH
group (n = 33) received prostaglandin F-two alpha (PGF, 0.150 mg), estradiol propionate (EP, 2 mg) and GnRH (50 µg) in 24 hour intervals (PGF at hour 0; EP at hour 24 and GnRH at hour 48). Cows in the hCG group (n = 30) were treated in a similar manner to the GnRH group, but, alternately, these cows received hCG (500 IU, i.m.) instead of GnRH. All cows in the treatment group were inseminated timely 16-20 hours after GnRH or hCG injections regardless of oestrus signs. During the study, animals exhibiting natural oestrus were inseminated 10-12 hours later and served as controls (n = 44). Cows in the hCG group have significantly higher synchronized (P = 0.018) and total (P < 0.05) ovulation rates and shorter intervals between the last hormone injection and ovulation (P = 0.012) compared to cows in the GnRH group. In both GnRH and hCG groups, cows with an active CL at the beginning of the treatment have acceptable pregnancy rates (40.0% and 47.6% respectively, P > 0.05). The PGF/EP/GnRH and PGF/EP/hCG treatments resulted in comparable pregnancy rates after TAI of lactating dairy cows at random stages of the oestrus cycle relating to those inseminated at natural oestrus (33.3%, 40.0% and 40.9% respectively, P > 0.05).
Medycyna Wet. 2008, 64 (2) 165 zed ovulations were obtained after estradiol
propio-nate (EP) and GnRH injections at the 48th and 60th hour,
respectively, following prostaglandin treatment at random stages of the oestrus cycle, and it has been suggested that this will make timed insemination possible (2).
The objectives of this study were to develop short oestrus synchronization methods allowing TAI in lactating dairy cows and to compare the effects of the use of hCG, instead of GnRH, on ovulation and pre-gnancy rates. An additional goal has been to determi-ne the effects of the presence or absence of an active corpus luteum (CL) on the treatment efficacy.
Material and methods
The experiment was conducted on a commercial dairy located in northwest Turkey where lactating Holstein cows were maintained in free-stall facilities, fed a totally mixed ration and milked twice daily. Mean daily milk production of the study cows was approximately 24 kg at the onset of the study. The experiment was conducted between Septem-ber and DecemSeptem-ber in 2006. All cows were under daily veterinary supervision. The first day of the treatments was considered as Day 0.
Sixty-three cows at random stages of the oestrus cycle on the 50th-95th postpartum day were randomly distributed
into two groups. Cows of GnRH group (n = 33) received PGF2a, (D-Cloprostenol 0.150 mg, i.m., Dalmazin, Vetaº, Turkey), Estradiol Propionate (EP, 2 mg, i.m., Akrofolline, Türfarma, Turkey) and GnRH (Lecirelin 50 µg, i.m., Dalmarelin, Vetaº, Turkey) at 24 hours intervals (PGF at hour 0; EP at hour 24 and GnRH at hour 48). Cows in the hCG group (n = 30) were treated in a similar manner to the GnRH group, but, alternately, these cows received hCG (500 IU, i.m., Pregnyl, Organon, Turkey) instead of GnRH. Transrectal ultrasound examinations were done by a single operator using a real-time, B-mode scanner with a 8.0-MHz linear-array transducer (Esaote Pie Medical, Falco, Nether-lands). Scannings were carried out daily for 6 days after the onset of treatments. Ovulation was taken as the sudden disappearance from one examination to the next
of a previously identified ovarian follicle ³ 8.5 mm in diameter. Cows that ovulated a follicle between 0 and 48 hours after GnRH or hCG injections were considered to have a synchronized ovulation. It was assumed that ovulation had failed in cows in which ovulation was not determined by the 72nd hour
post-GnRH or hCG injection. Regardless of oestrus signs, all cows in the treatment group were inse-minated timely 16-20 hours after GnRH or hCG injections.
Blood samples were obtained from the coccy-geal vein of cows in treatment groups at the start of the treatment (Day 0). Serum was harvested, frozen and stored in plastic tubes at 20°C until analyzed. The concentration of progesterone (P4) in the serum was measured in all samples by radioimmunoassay using a commercial kit (DSL 3900, Progesterone, DSL, USA). The sensitivity of the assay was 0.14 ng/ml. Cows in treatment
groups were grouped into CL-classes retrospectively ac-cording to plasma P4 concentrations of the blood samples collected immediately before PGF injections. A concen-tration higher than 1 ng/ml was considered to indicate the presence of a functional CL.
During the study, animals exhibiting natural oestrus within the corresponding 50 to 96 days after parturition were inseminated and served as controls (n = 44). Animals were inspected for signs of oestrus in the early morning and late afternoon for 30 to 45 minute. Oestrus was confir-med by rectal examination at the time of insemination. Control cows suspected to be in natural oestrus were inse-minated 10-12 h later. Pregnancy diagnosis was performed by ultrasound 35 to 40 days post-insemination.
Statistical Analysis. Categorical data such as pregnan-cy rates and percentage of cows detected in oestrus were evaluated for treatment differences by Chi-square. Quanti-tative data such as interval from last PGF injection to oestrus and plasma P4 concentrations were evaluated by indepen-dent samples t-test. All statistical analyses were carried out using the SPSS program v. 10.0 for Windows (SPSS Inc, Chicago, IL). Means and standard deviations are presented in tables.
Results and discussion
The incidence of oestrus, the interval of PGF injec-tion to oestrus, and the size of ovulatory follicles on the last hormone injection day in cows in the GnRH and hCG groups were found to be very similar (P > 0.05). Cows in the hCG group have significantly higher synchronized (P = 0.018) and total (P < 0.05) ovulation rates and shorter intervals between the last hormone injection (GnRH or hCG) and ovulation (P = 0.012) compared to cows in the GnRH group. In addi-tion, pregnancy rates in cows in the hCG group were found to be slightly higher than those of the GnRH group (P > 0.05, tab. 1).
Both GnRH (76.0% vs. 37.5%, P < 0.05) and hCG group (85.7% vs. 33.3%, P < 0.01), cows with active CL at the beginning of the experiment exhibited
signi-s p u o r G (nG=nR3H3) (nh=CG30) P ) % ( y d u t s e h t f o t e s n o e h t t a L C e v it c A 75.8 70.0 P>0.05 ) % ( e c n e d i c n i s u rt s e O 66.7 70.0 P>0.05 ) % ( s e t a r n o it a l u v O n o it c e j n i G C h r o H R n G r e tf a h 4 2 o t 0 13.0 30.0 P<0.01 d e zi n o r h c n y S 27.3 56.7 P=0.018 l a t o T 63.6 86.7 P<0.05 ) % ( e t a r y c n a n g e r P 33.3 40.0 P>0.05 H R n G e h t n o s e l c il l o f y r o t a l u v o f o e zi S ) m m ( y a d n o it c e j n i G C h r o 14.9±4.69 16.7±5.14 P>0.05 ) h ( l a v r e t n i s u rt s e o o t n o it c e j n i F G P 46.4±4.22 48.1±2.10 P>0.05 ) h ( l a v r e t n i n o it a l u v o o t G C h r o H R n G 48.6±14.44 35.1±19.77 P=0.012
Tab. 1. Comparison of reproductive performance of cows in the GnRH and hCG groups
Medycyna Wet. 2008, 64 (2) 166
ficantly higher oestrus rates than cows without active CL. In the hCG group, the synchronized ovulation rates in cows with and without a functional CL at the beginning of the study were found to be similar (P > 0.05), while in the GnRH group, the synchronized ovulation rates in cows with an active CL were found to be significantly higher (36.0% vs. 0.0%, P < 0.05). In both GnRH (40.0% vs. 12.5%) and hCG (47.6% vs. 22.2%) groups, pregnancy rates in cows with an active corpus luteum were found to be higher, but the difference between them was determined to be stati-stically insignificant (P > 0.05, tab. 2). Similar pre-gnancy rates were obtained in the GnRH and hCG group in comparison to cows inseminated first at the natural oestrus (33.3%, 40.0% and 40.9%, GnRH, hCG and control groups respectively, P > 0.05, tab. 3).
Previous short oestrus synchronization studies have mostly been conducted on cows with a determinable CL (5-7). In this study we initiated the treatments at random stages of the oestrus cycle. In addition, we evaluated the treatment efficacy in cows with and without an active CL at the beginning of the trial, by classifying cows retrospectively according to their serum P4 concentrations. In the hCG group, synchro-nized ovulation rates of cows with and without a func-tional CL at the beginning of the study were found to be similar, while in the GnRH group, synchronized ovulation rates of cows with an active CL were dis-covered to be significantly higher (36% vs. 0.0%, P < 0.05). Although the pregnancy rates of cows with an active CL at the beginning of the experiment were found rather high in both GnRH (40.0% vs. 12.5%) and the hCG group (47.6% vs. 22.2%), the difference
between them was determined to be stati-stically insignificant. The reason for this is probably the low number of cows not having an active CL in both groups. Meantime, the results of our study clearly indicate that in cows with an active CL at the beginning of the experiment, the treatment will result in acceptable pre-gnancy rates in both groups. It has been reported that pregnancy rates in cows are positively associated with higher proge-sterone concentration before AI (4).
Although both treatments were initia-ted at random stages of the oestrus cycle in our study, cows in the hCG group had significantly higher synchronized and total ovulation rates, and a shorter inter-val between the last hormone injection and ovulation, compared to cows in the GnRH group. This fact can be explained by the stronger and longer LH--effect potential of hCG than GnRH (3) and also force us consider that estradiol may have a more synergistic effect with hCG, rather than GnRH. Although it is statistically insignificant, cows in the hCG group had higher pregnancy rates than those of the GnRH group. This difference can be explained by the higher total and synchronized ovulation rates of the hCG group. In 9 of 30 cows (30.0%) in the hCG group, the ovula-tions were seen before the TAI. Only one of these cows conceived (11.1%), while cows ovulated after TAI had a pregnancy rate of 52.4% (11/21). These data imply that if TAI is applied at an earlier stage (i.e. 6-8 hours after hCG), treatment in the hCG group may result in more favourable pregnancy rates. It is known that lu-teal development following insemination is effective for the maintenance of pregnancy. It has been reported that administration of hCG induces a greater increase in plasma progesterone concentration compared to GnRH treatment (12). In our study luteal development following TAI was not investigated. However, when combined with data from literature, ovulation and part-ly the pregnancy rates in our study suggest that use of hCG instead of GnRH 48 hours after PGF treatment in short synchronization programs can increase treat-ment efficacy. In order to prove this hypothesis, new studies including a large number of animals are re-quired.
Many researchers accept the fact that follicular development should also be synchronized prior to synchronization of luteolysis with PGF. This is likely the reason why only a limited number of studies have been conducted on short synchronization methods. Although many programs have been developed for timed insemination, the stage of the oestrus cycle at the beginning of treatments affects pregnancy rates significantly (9). Therefore, there have recently been efforts to develop pre-synchronization methods in order to adjust the cycle stage at the beginning of
Tab. 2. Reproductive performances of cows with or without an active CL at the beginning of the experiment in GnRH and hCG groups
Explanations: The difference between the sub-groups of each main group is signi-ficant relating the traits at the same row (* P < 0.05, ** P < 0.01)
s p u o r G GnRH hCG y d u t s e h t f o t e s n o e h t t a L C e v it c A (nN=o8) (nY=e2s5) (nN=o9) (nY=es21) e h t n o s e l c il l o f y r o t a l u v o f o e zi S ) m m ( y a d n o it c e j n i H C h r o H R n G 12.4±4.56 15.7±4.52 16.6±3.91 16.8±5.68 ) % ( e c n e d i c n i s u rt s e O 37.5* 76.0* 33.3** 85.7** ) % ( e t a r y c n a n g e r P 12.5* 40.0* 22.2** 47.6** ) % ( s e t a r n o it a l u v O G C h r o H R n G r e tf a h 4 2 o t 0 0.0* 14.0* *55.6* *19.0* d e zi n o r h c n y S 0.0* 36.0* 55.6 57.1 l a t o T 50.0** 68.0* 77.8 90.5 p u o r G (nGn=R3H3) (nh=CG30) (Cno=n4rto4l) P ) % ( I A t s ri f o t e t a r y c n a n g e r P 33.3 40.0 40.9 P>0.05
Tab. 3. First insemination pregnancy rates of cows in GnRH, hCG and control groups
Medycyna Wet. 2008, 64 (2) 167 synchronization treatments to a desired period (1, 8).
Reproductive efficiency in dairy cattle has declined over the last five decades. Conception rates were ap-proximately 55% in porous dairy cows in the 1950s, while current rates are approximately 35% or lower for timed insemination (13). In order to obtain higher pregnancy rates, gradually more and more researchers consider that different treatments should be used at dif-ferent periods of the cycle, following the determina-tion of the oestrus period of cows (9). Despite its high error margin, rectal examination has been used widely in the past for the determination of cows with an active CL. Recently, use of ultrasound and practice milk or blood progesterone measurement tests for this purpose allow very accurate determinations. Our results confirm the observations that short oestrus synchronization treatments result in acceptable pre-gnancy rates in lactating dairy cows with a determina-ble CL as reported previously (5-7). One of the best methods in evaluating the success of synchronization treatments is the comparison of the pregnancy rates with the rates of inseminations in natural oestruses. Our data also showed that the PGF/EP/GnRH and PGF/ EP/hCG treatments resulted in comparable pregnancy rates after timed AI of lactating dairy cows at random stages of the oestrus cycle relating to those insemina-ted at natural oestrus. Moreover, the results of the pre-sent and previous studies indicate that cows without an active CL at the onset of the study will cause a de-crease in the total pregnancy rates of a group of cows subjected to short synchronization treatments. Cows having < 1 ng/ml plasma P4 concentrations include those in metoestrus, proestrus or oestrus of the cycle and also those in the anoestrus, if any. Cirit et al. (2) have reported that although the short oestrus synchro-nization treatment including PGF injection at hour 0, EP at the 48th hour, and GnRH at the 60th hour resulted
in ovulations in 3 of 5 anoestrus cows, there was no functional CL development following the ovulation in 2 of these 3 cows, and there was no pregnancy fol-lowing TAI 16-20 hours after the GnRH injection in the cows at a late metoestrus period. In an earlier study we determined that an additional estradiol pro-pionate injection on day 8 of the ovsynch protocol increased the pregnancy rates numerically (42.9% vs. 63.6%, P > 0.05) in primiparous dairy cows without an active CL at the onset of the study (1). This treat-ment resembles mostly the PGF/EP/GnRH and PGF/ EP/hCG treatments in the present study aside from the first GnRH injection. Therefore, to obtain better pre-gnancy rates in total by the short oestrus synchroniza-tion treatments, a different strategy can be employed. At any day, approximately 60% of non-pregnant cyclic cows will be expected to have an active CL. These cows can receive the PGF/EP/hCG treatment, and the synchronization can be initiated with a GnRH injec-tion followed by the PGF/EP/hCG treatment 7 days later in cows without a functional CL. This enables
most of a group of cows to be inseminated sooner with lower hormone expenses and no oestrus detection. Since a GnRH injection 7 days prior to the treatment synchronizes the follicular development, PGF/EP/hCG treatment can yield higher pregnancy rates in cows without an active CL.
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