Replacement of the First GnRH Administration in the Ovsynch Protocol by Selecting Cows

Hartman P. , J . Zaj íc , S. âech, Z. Jan, R. DoleÏel : Replacement of the First GnRH Administration in the Ovsynch Protocol by Selecting Cows According to the Stage of Follicular Development. Acta Vet. Brno 2006, 75: 65-72. The aim of the study was to replace the first GnRH in the Ovsynch protocol by selecting cows bearing corpus luteum as well as follicles in a defined stage of development at PGF2α administration. Additionally, various terms of GnRH administration after PGF2α were tested. Seventy five non-pregnant cows bearing corpus luteum were divided into groups according to the phase of follicular development on D 0 (day of PGF2α administration) – growth (GR, follicles 3.0 – 7.9 mm in diameter), early dominance (ED, dominant follicle 8.0 – 14.9 mm) and late dominance (LD, dominant follicle 15.0 – 23.0 mm). In addition, the cows were divided into groups according to the terms of GnRH administration (24, 48 or 72 h after PGF2α). In this way, groups GR 48 (n = 5), GR 72 (n = 6), ED 24 (n = 10), ED 48 (n = 12), ED 72 (n = 12), LD 24 (n = 10), LD 48 (n = 10) and LD 72 (n = 10) were established. Growth of ovulatory follicle, term of ovulation, insemination and conception rates as well as relation of the size of preovulatory follicle (day of ovulation) to the size of following corpus luteum (day 14) were evaluated. The highest level of synchronization of ovulation (100% on D 3) as well as conception rate (50%) was achieved in group ED 48. This protocol gives an opportunity of timing artificial insemination to 18 – 24 hours after GnRH administration, as ovulation occurs with a high probability within 24 48 hours after GnRH administration. Oestrus synchronization, follicle diameter, ovulation, timing of insemination Synchronization of oestrus in cows with a standard term of ovulation provides a good opportunity for the timing of artificial insemination. It can decrease losses caused by poor oestrus detection and in addition, it can improve the embryo-transfer efficiency. The term of ovulation after administration of PGF2α in the luteal phase of the oestrous cycle is variable because it depends on the stage of follicular development at the time of treatment (King et al. 1982; Macmil lan and Henderson 1984; Kastel ic et al. 1990; Savio et al. 1990; Kastel ic and Ginther 1991). Follicular development can be synchronized by administration of GnRH, followed by removal of the dominant follicle (ovulation or atresia) and recruitment of a new follicular wave (Thatcher et al. 1989; Macmil lan and Thatcher 1991; Twagiramungu et al. 1995). This effect of GnRH is applied in the Ovsynch protocol (Pursley et al. 1995) as well as similar methods of oestrus synchronization in cows (Pursley et al. 1997; Pancarci et al. 2002; Thatcher et al. 2002). Nevertheless, the ovulation of a premature follicle after the first GnRH administration (Roy and Twagiramungu 1999; DeJarnet te et al. 2001) and an incomplete luteal regression following PGF2α (Burke et al. 1996; Moreira et al. 2000) are described. This is probably associated with the conception failure in the Ovsynch protocol (Kim et al. 2003). In our study we replaced the first GnRH in the Ovsynch protocol by selecting cows bearing corpus luteum as well as follicles in a defined stage of development at PGF2α administration. In addition, the various terms of GnRH administration after PGF2α were tested. ACTA VET. BRNO 2006, 75: 65–72 Address for correspondence: Doc. MVDr. Radovan DoleÏel, CSc. Clinic of Diseases of Ruminants University of Veterinary and Pharmaceutical Sciences Palackého 1-3, 612 42 Brno, Czech Republic Phone: +420 541 562 316 E-mail: dolezelr@vfu.cz http://www.vfu.cz/acta-vet/actavet.htm Materials and Methods Experimental animals Seventy five lactating cows (crossbreeds Bohemian Pied Cattle and Holstein) bearing corpora lutea (CLs) within 55 and 100 days post partum were used in our study. They were housed in three commercial dairy farms, and the average milk production of the herds was between 6 500 and 8 000 kg of milk per lactation. Conception rates after the first insemination in the herds were around 35 40% during the period of the study. The cows were divided into groups according to the phase of follicular development in the ultrasonographical image – growth (GR, follicles 3.0 – 7.9 mm in diameter, n = 11), early dominance (ED, dominant follicle 8.0 – 14.9 mm, n = 34) and late dominance (LD, dominant follicle 15.0 – 23.0 mm, n = 30). A follicle ≥ 8 mm in diameter at least 2.0 mm larger than the remaining ones was considered as the dominant follicle. The cows were treated by synthetic analog of PGF2α cloprostenol (Oestrophan, Bioveta, Ivanovice, Czech Rep., 500 μg pro toto, i.m.) and following administration of GnRH (lecirelinum, Supergestran, Ferring-Leciva, Jesenice, Czech Rep., 100 μg pro toto, i.m.) were performed 24, 48 or 72 h after PGF2α. In addition, the cows were divided in groups according to the terms of GnRH administration. In this way groups GR 48 (n = 5), GR 72 (n = 6), ED 24 (n = 10), ED 48 (n = 12), ED 72 (n = 12), LD 24 (n = 10), LD 48 (n = 10) and LD 72 (n = 10) were established. Group GR 24 was not established because of a small number of cows bearing follicles in the growth phase of the follicular wave. Observat ion Transrectal ultrasonographical examinations (Aloca SSD 500 with a 5 MHz rectal probe, Tokyo, Japan) of ovarian structures were performed daily from day 0 (day of PGF2α administration) to day 5 and on day 14. Ultrasonographical images of ovarian structures were taken by a digital video camera (Sony DCR-TRV 235 E) and data were compiled in the studio DV plus Pinnacle® system. Additional measurements of ovarian structures were made in Auto-CAD® 2002. Diameters of ovarian structures were determined as an arithmetical mean of two axes vertical to each other. Peripheral blood samples were obtained on days 0, 2 and 14. The blood was centrifuged and it was stored at – 20 °C until progesterone determination by RIA, using commercial sets (Immunotech, Prague, Czech Republic). The detection of oestrus was performed by herd personnel twice a day and cows in oestrus were inseminated. Pregnancy diagnosis was performed ultrasonographically on day 25 and 39 after insemination (see Fig. 1). Disappearance of the dominant follicle followed by appearance of corpus luteum in the same place of the ovary on day 14 was considered as evidence of ovulation. Oestrus was considered as synchronized when ovulation appeared up to day 5 after PGF2α administration. Evaluat ion Growth of the ovulatory follicle, the term of ovulation, insemination and conception rates as well as the relation of the size of the preovulatory follicle (day of ovulation) to the size of the following corpus luteum (day 14) were evaluated. A comparison was made of follicle sizes within an individual group as well as among the groups, using parametric tests. Statistical analysis of ovulation occurrence on day 3 (day 0 – day of PGF2α administration) was done on one hand among common groups divided according to the stage of follicular development at PGF2α administration, regardless of the term of GnRH administration (groups GR, ED and LD); and on the other hand it was done among groups divided according to the term of GnRH administration, regardless of the stage of follicular development at PGF2α administration (groups 24, 48 and 72). Synchronizations of ovulation were evaluated using a Two-way table, which is available at the web site: http://caspar.bgsu.edu/~software/java/1Contingency.html. This table used Chi-square (χ2) or G-tests (G). Statistically significant deviation is evaluated by the Freeman-Tukey test which determines unexpected higher or lower values in the frame of a null hypothesis. Willam’s correction was performed and all statistics were compared by the Chi-square distribution with appropriate degrees of freedom. The relationship of the sizes of preovulatory follicles to the sizes of corpora lutea was evaluated by Kruskal-Wallis or Steel-Dwass tests. 66 Fig. 1. Scheme of the study

Synchronization of oestrus in cows with a standard term of ovulation provides a good opportunity for the timing of artificial insemination.It can decrease losses caused by poor oestrus detection and in addition, it can improve the embryo-transfer efficiency.The term of ovulation after administration of PGF 2α in the luteal phase of the oestrous cycle is variable because it depends on the stage of follicular development at the time of treatment (King et al. 1982;Macmillan and Henderson 1984;K astelic et al. 1990;Savio et al. 1990;Kastelic and G in ther 1991).Follicular development can be synchronized by administration of GnRH, followed by removal of the dominant follicle (ovulation or atresia) and recruitment of a new follicular wave (Thatcher et al. 1989;Macmillan and Thatcher 1991;Twagiramungu et al. 1995).This effect of GnRH is applied in the Ovsynch protocol (Pursley et al. 1995) as well as similar methods of oestrus synchronization in cows (Pursley et al. 1997;Pancarci et al. 2002;Thatcher et al. 2002).Nevertheless, the ovulation of a premature follicle after the first GnRH administration (Roy and Twagiramungu 1999;DeJarnette et al. 2001) and an incomplete luteal regression following PGF 2α (Burke et al. 1996;Moreira et al. 2000) are described.This is probably associated with the conception failure in the Ovsynch protocol (K im et al. 2003).In our study we replaced the first GnRH in the Ovsynch protocol by selecting cows bearing corpus luteum as well as follicles in a defined stage of development at PGF 2α administration.In addition, the various terms of GnRH administration after PGF 2α were tested.

Experimental animals
Seventy five lactating cows (crossbreeds Bohemian Pied Cattle and Holstein) bearing corpora lutea (CLs) within 55 and 100 days post partum were used in our study.They were housed in three commercial dairy farms, and the average milk production of the herds was between 6 500 and 8 000 kg of milk per lactation.Conception rates after the first insemination in the herds were around 35 -40% during the period of the study.The cows were divided into groups according to the phase of follicular development in the ultrasonographical image -growth (GR, follicles 3.0 -7.9 mm in diameter, n = 11), early dominance (ED, dominant follicle 8.0 -14.9 mm, n = 34) and late dominance (LD, dominant follicle 15.0 -23.0 mm, n = 30).A follicle ≥ 8 mm in diameter at least 2.0 mm larger than the remaining ones was considered as the dominant follicle.The cows were treated by synthetic analog of PGF 2α cloprostenol (Oestrophan, Bioveta, Ivanovice, Czech Rep., 500 µg pro toto, i.m.) and following administration of GnRH (lecirelinum, Supergestran, Ferring-Leciva, Jesenice, Czech Rep., 100 µg pro toto, i.m.) were performed 24, 48 or 72 h after PGF 2α .In addition, the cows were divided in groups according to the terms of GnRH administration.In this way groups GR 48 (n = 5), GR 72 (n = 6), ED 24 (n = 10), ED 48 (n = 12), ED 72 (n = 12), LD 24 (n = 10), LD 48 (n = 10) and LD 72 (n = 10) were established.Group GR 24 was not established because of a small number of cows bearing follicles in the growth phase of the follicular wave.

Observation
Transrectal ultrasonographical examinations (Aloca SSD 500 with a 5 MHz rectal probe, Tokyo, Japan) of ovarian structures were performed daily from day 0 (day of PGF 2α administration) to day 5 and on day 14.Ultrasonographical images of ovarian structures were taken by a digital video camera (Sony DCR-TRV 235 E) and data were compiled in the studio DV plus Pinnacle ® system.Additional measurements of ovarian structures were made in Auto-CAD ® 2002.Diameters of ovarian structures were determined as an arithmetical mean of two axes vertical to each other.Peripheral blood samples were obtained on days 0, 2 and 14.The blood was centrifuged and it was stored at -20 °C until progesterone determination by RIA, using commercial sets (Immunotech, Prague, Czech Republic).The detection of oestrus was performed by herd personnel twice a day and cows in oestrus were inseminated.Pregnancy diagnosis was performed ultrasonographically on day 25 and 39 after insemination (see Fig. 1).Disappearance of the dominant follicle followed by appearance of corpus luteum in the same place of the ovary on day 14 was considered as evidence of ovulation.Oestrus was considered as synchronized when ovulation appeared up to day 5 after PGF 2α administration.

Evaluation
Growth of the ovulatory follicle, the term of ovulation, insemination and conception rates as well as the relation of the size of the preovulatory follicle (day of ovulation) to the size of the following corpus luteum (day 14) were evaluated.
A comparison was made of follicle sizes within an individual group as well as among the groups, using parametric tests.Statistical analysis of ovulation occurrence on day 3 (day 0 -day of PGF 2α administration) was done on one hand among common groups divided according to the stage of follicular development at PGF 2α administration, regardless of the term of GnRH administration (groups GR, ED and LD); and on the other hand it was done among groups divided according to the term of GnRH administration, regardless of the stage of follicular development at PGF 2α administration (groups 24, 48 and 72).Synchronizations of ovulation were evaluated using a Two-way table, which is available at the web site: http://caspar.bgsu.edu/~software/java/1Contingency.html.This table used Chi-square (χ 2 ) or G-tests (G).Statistically significant deviation is evaluated by the Freeman-Tukey test which determines unexpected higher or lower values in the frame of a null hypothesis.Willam's correction was performed and all statistics were compared by the Chi-square distribution with appropriate degrees of freedom.The relationship of the sizes of preovulatory follicles to the sizes of corpora lutea was evaluated by Kruskal-Wallis or Steel-Dwass tests.

Results
The growth of ovulatory follicles is shown in Tables 1, 2 and 3. Significant differences in follicle diameters were found between follicles on day of PGF 2α administration and follicles on day of GnRH administration in GR, LD (p < 0.05) as well as ED (p < 0.01) groups.The differences were not proved between follicles on the day of GnRH administration and follicles on the day of ovulation.Even follicles became smaller in LD groups during this period.
Ovulation was observed in 72% (54/75) of all experimental cows up to day 5. Ovulations occurred on days 2, 3 and 4. The highest incidence of ovulation was found on day 3.For this reason we evaluated ovulations which occurred on day 3.The highest level of synchronization of ovulation was found in group ED 48 in which 100% (12/12) cows ovulated on day 3 after PGF 2α administration on day 0 (Table 4).
In common groups divided according to the stage of follicular development at PGF 2α administration (regardless to term of GnRH administration) the highest occurrence of ovulation on day 3 (the 3 th day after PGF 2α administration) was found in group ED and the lowest occurrence was found in group GR (p < 0.01), see Fig. 2.
On the other hand, in common groups divided according to the term of GnRH

67
Table 1.Means (± SEM) of preovulatory follicle diameters in groups GR 48 (n = 3) and GR 72 (n = 1) (mm) Table 4. Occurrence of ovulation in all experimental groups up to day 5 after PGF 2α administration on day 0 administration (regardless of the stage of follicular development at PGF 2α administration), the highest occurrence of ovulation on day 3 was found in group 72 and the lowest occurrence was found in group 24 (p < 0.05), see Fig. 3.
The highest occurrence of ovulation, within 5 days after PGF 2α administration, was found in group 48 (p < 0.001).
Insemination was done in 45% (34/75) experimental cows and the average pregnancy rate was only 24%.The highest conception rate (50%) was found in group ED 48.Positive correlations were found between sizes of preovulary follicles on the day of ovulation and sizes of corpora lutea on day 14 in groups ED 24, ED 48, ED 72, LD 24 and LD 72 (p < 0.001) as well as in groups GR 48 and LD 48 (p < 0.01).Group GR 72 could not be evaluated for a small number of evaluated cows.

Discussion
We could not establish complete groups of cows bearing only small follicles (3.0 -7.9 mm in diameter) on ovaries.Low incidence of these cows was probably caused by frequent persistence of a morphologically dominant follicle during the growth phase of a new follicular wave.Persistence of morphological dominance after the loss of functional dominance in follicles during follicular waves in cows has been described (Fortune 1993; Savio et al. 1990).For that reason the growth phase of a follicular wave cannot be exactly differentiated from late dominance in cows on the basis of ultrasonographical examination.Fig. 2. Terms of ovulation in experimental groups (24, n=18; 48, n=20, 72, n=16) in relationship to phase of follicular development on D 0 (median, max.-min).Thus some cows bearing a follicle 15.0 -23.0 mm in diameter could be in fact in the growth phase of a new follicular wave in our study.There is a chance to induce a growth phase of a new follicular wave artificially, by puncturing follicles under ultrasonographical guidance (the ovum pick up method -OPU).However, we did not use it, as the OPU may negatively influence the next oestrous cycle.Smaller corpora lutea with lower progesterone concentrations in peripheral blood and a low conception rate after OPU were described (Vasconcelos et al. 2001).
We found high growth potency of ovulatory follicles after PGF 2α up to GnRH administration in all experimental groups.Low growth potency after GnRH up to ovulation especially in groups LD is partly in agreement with authors DiZerega and Hodgen (1980) who found in cows treated by GnRH an outstandingly higher growth potency of small growing follicles before selection, compared to large dominant follicles.Reduction of size in some preovulatory follicles can be caused by a decrease of follicle tension after a temporal decrease of FSH concentration (G in th er 2000).
The term of ovulation after single administration of PGF 2α in cows bearing corpus luteum is variable, because it depends on the stage of follicular development at the time of treatment (Macmillan and Henderson 1984;Savio et al. 1990;Kastelic et al. 1990;Turzillo and Fortune 1993).The highest dispersion of ovulation after PGF 2α was described when treatment was performed in the middle of the oestrous cycle (Tanabe and Hann 1984).Nevertheless, most cows ovulate on day 3 after treatment (Stellflung et al. 1975;K rál et al. 1981;Larson and Ball 1992).In our study, 72% of cows ovulated up to day 5 after PGF 2α administration regardless of the stage of follicular development at PGF 2α treatment as well as the term of GnRH administration.In agreement with the above stated authors, most cows (55.6%) ovulated on day 3 after treatment.The highest level of synchronization of ovulation after PGF 2α was found when treatment was performed on cows bearing corpus luteum as well as an early dominant follicle, and GnRH was administered 48 hours after PGF 2α (group ED 48).Ovulation occurred in 100% of these cows on day 3 after PGF 2α treatment.
Although a relatively standard occurrence of ovulation after PGF 2α treatment was found in our study, a few cows (45%) were inseminated and the total conception rate was very low (24%).Ten cows were inseminated despite ovulation not having occurred.On the other hand, 30 cows were not inseminated despite ovulation having occurred.These data show low efficiency of oestrus detection in the herds.A poor rate of oestrus detection in herds of milk cows is also described by many other authors (Noakes et al. 2001;Arney et al. 1994;Kerbrat a Disenhaus 2004).The authors mention the level of incorrect detection around 61%.An especially low rate of oestrus detection was found after PGF 2α treatment (Slenning and Farver 1990).For this reason, the low conception rate was partly caused by poor oestrus detection allowing insemination of unovulatory cows in our study.On the basis of these data, a higher conception rate after insemination could be expected regardless of oestrus detection.A low conception rate is often associated with abnormal corpus luteum with short and low production of progesterone after insemination (Rosenberg et al. 1990;G yawu et al. 1991;M o re ll et al. 1991;Xu et al. 1997).Nevertheless, the quality of corpus luteum depends on the quality of the preovulatory follicle (Smith 1986;Vasconcelos et al. 2001).Likewise, the length as well as the level of higher concentration of progesterone before ovulation influences the quality of preovulatory follicles as well as the quality of the subsequent corpus luteum (White et al. 1985;Smith 1986;Vasconcelos et al. 2001).For this reason low conception rate could be partly caused by the short luteal phase before ovulation in our study.Generally, environmental factors significantly influence the conception rate.But the conception rate after the first insemination in the herds involved in the study was higher (35 -40%) at the time of the experiment.These data show that a low conception rate is probably not caused by environmental conditions.Frequent manipulation with uterus and ovaries within the transrectal ultrasonographical examination could negatively influence conception, too.Roelofs et al. (2004) reported that frequent ultrasonographical examination did not have a negative effect on oestrus behaviour nor the rate of oestrus detection.But higher variability of the term of ovulation was noted.Nevertheless, the relatively high conception rate (50%) in group ED 48 shows that the main factors influencing conception rate in our study are probably the protocol of hormonal treatment as well as the quality of ovarian structures.Lower conception rate (31.1%) was found after the Ovsynch protocol (Peters and Pursley 2003).
Our results show that PGF 2α treatment in cows bearing corpus luteum as well as the early dominant follicle with following administration of GnRH 48 hours after PGF 2α represents an efficient protocol of oestrus synchronization.This protocol gives an opportunity for timing artificial insemination to 18 -24 hours after GnRH administration because ovulation occurs with a high probability within 24 -48 hours after GnRH administration.