Effect of GnRH Administration on Holstein Cows with Follicular Cysts or Ovarian Quiescence as Determined by Ultrasonography at Regular Checkup

Changes in the ovaries were monitored every 2 to 3 weeks using real-time ultrasonography as part of regular reproductive checkups in 178 multiparous and 109 heifer (total 287) cows which had been diagnosed as having follicular cysts (77 cows) or ovarian quiescence (210 cows) and treated once with gonadotropin-releasing hormone, GnRH (fertirelin acetate at 100 μg i.m.). Following the one-time treatment, 96 cows (33.4%) showed estrus and underwent artificial insemination by the next reproductive checkup (Cow AI), 87 cows (30.3%) were given dinoprost as prostaglandin F2α (PG) to induce estrus at the next checkup because of existence of Corpus Luteum (CL) in their ovaries (Cow PG) and there was no change (no effect) in the ovaries of 104 cows (Cow NE, 36.2%). The number of cows which conceived during the estrus cycle following injection of GnRH was 39 (40.6%) in Cow AI and 38 (43.7%) in Cow PG. In conclusion, one-time GnRH administration to cattle with ovarian cysts or quiescence but without CL at a regular reproductive checkup may effectively shorten the time to conception.


INTRODUCTION
In reproductive management for cows, to improve reproductive performance it is important to detect and treat abnormalities in reproductive organs as soon as possible.Introduction of the use of portable transrectal real-time ultrasonography into reproductive management programs has enabled visualization of ovarian pathologies that are not accurately detected via rectal palpation (Fricke, 2002).Ovarian quiescence and follicular cysts are the most common ovarian disturbances in cows or heifers failing to show signs of estrus after parturition or after reaching breeding age, respectively (Bosu and Peter, 1987;Opsomer et al., 1998).Although gonadotropin-releasing hormone (GnRH) has been shown to be effective for the treatment of ovarian quiescence and follicular cysts (Isobe et al., 2007;Mollo et al., 2012;Peter, 2004), there are no reports showing the effectiveness of GnRH administration in cows with ovarian quiescence or follicular cysts diagnosed by ultrasonographic imaging at regular reproductive checkups.Furthermore, although there are many treatment strategies for ovarian quiescence or follicular cyst, adaptating plural remedies at reproductive checkups in the field may complicate conducting these checkups and evaluation of the effects of these treatments.The objective of this study was to investigate the effect of GnRH given to cows upon diagnosis based on ultrasonographic images at regular reproductive checkups with the aim of improving reproductive performance.(multiparous, 178; nulliparous, 109; total, 287) on 17 dairy farms which were conducting regular reproductive checkups including ultrasonographic examination were included in the study.The multiparous cows had not shown sign of estrus at 30 days after parturition and the nulliparous cows had not shown the first estrus by attaining the age of 13 months.

Cows
Changes in the ovaries were monitored every 2 to 3 weeks using real-time ultrasonography (50S Tringa equipped with a 7.5-MHz transrectal linear transducer; Sumire Medical Co., Ltd., Tokyo, Japan).Cows were administrated GnRH (fertirelin acetate 100 μg i.m.) if corpus luteum (CL) was not detected at two consecutive examinations.If large follicles (diameter>2.5 cm) were detected, the cows were diagnosed as having a follicular cyst (Group FC) and others were diagnosed as showing ovarian quiescence.Furthermore the cows with ovarian quiescence were allocated to Group FOQ (with follicles having a diameter of 8 mm or greater) and Group OQ (with follicles less than 8 mm).2) Group FC: cows which had cystic follicles at GnRH administration; Group FOQ: cows which had follicle ≥8 mm at GnRH administration; Group OQ: cows which had follicle <8 mm at GnRH administration; Cow AI: cows which showed estrus after GnRH administration and received artificial insemination by the next reproductive checkup; Cow PG: cows which were given prostaglandin F 2α at the next reproductive checkup; Cow NE: cows in which there was not any change in their ovaries After GnRH administration, all cows were classified into three groups based on the response to GnRH.The Cow AI group included cows that showed estrus and had undergone Artificial Insemination (AI) by the next reproductive checkup, The Cow PG group was given prostaglandin F 2α (PG) (Pronalgon F, Pfizer, Tokyo, Japan) at the next reproductive checkup based on the presence of CL and no changes were observed in the ovaries of the Cow NE group.The GnRH treatment was considered to have been effective in the Cow AI and Cow PG groups.The incidence of cows showing estrus within 16 days after GnRH administration was compared among Group FC, Group FOQ and Group OQ.Conception was confirmed by ultrasonography at 41 days or later after artificial insemination.

RESULTS
At the time of GnRH administration, 77 cows (26.8%) belonged to Group FC, 166 cows (57.8%) belonged to Group FOQ and 44 cows (15.3%) belonged to Group OQ (Table 1).After GnRH administration, 96 cows (33.4%) showed estrus and underwent artificial insemination before the next reproductive checkup (Cow AI), 87 cows (30.3%) were given PG at the next reproductive checkup due to the presence of CL in their ovaries (Cow PG) and there were no changes in the ovaries of 104 cows (36.2%) (Cow NE) (Table 1).The number of cows in which GnRH was effective (Cow AI+Cow PG) was 51 (66.2%) in Group FC, 109 (65.7%) in Group FOQ and 23 (52.3%) in Group OQ and there was no significant difference among the groups (Table 1).The number of cows in the Cow AI and Cow PG groups was 22 (28.6%) and 29 (37.7%) in Group FC, 62 (37.3%) and 47 (28.3%) in Group FOQ and 12 (23.7%)and 11 (25.0%) in Group OQ, respectively (Table 1).The number of days from GnRH administration to artificial insemination was 15.0±6.4days in the Cow AI group and 23.0±8.7 days in the Cow PG group and it was significantly shorter in the Cow AI group than in the Cow PG group (p<0.01)(Table 2).In the Cow AI and Cow PG groups, 39 (40.6%) and 38 (43.7%) cows, respectively, conceived and there was no significant difference between the two groups (Table 2).Of 183 cows in which effectiveness of GnRH was confirmed, 60 cows (32.8%) underwent AI within 16 days of GnRH administration.The distribution of these cows in each group was 12 (23.5%) in Group FC, 39 (35.8%) in Group FOQ and 9 (39.1%) in Group OQ, respectively and there were no significant differences among the groups.

DISCUSSION
As a result of GnRH administration, 96 cows (33.4%) showed estrus and underwent AI by the time of the next reproductive checkup (Cow AI) and 87 cows (30.3%) showed CL in the ovaries at the next reproductive checkup and received PG (Cow PG).This shows that the GnRH administration was effective in 183 cows (63.8%).The number of days from GnRH administration to artificial insemination was significantly shorter in the Cow AI group than in the Cow PG group.Because the dominant follicle needed to be ovulated before PG was given in the Cow PG group, it might have taken longer to show estrus.Mollo et al. (2012) reported a cure rate of follicular cysts by GnRH of 64.3% and the effective rate of GnRH administration in Group FC (66.2%) was similar.When GnRH is given to cows with follicular cysts, a smaller follicle coexisting with cystic follicle tends to ovulate and form the CL instead of the cystic follicle (Isobe et al., 2007).In this study, 22 out of 77 cows in Group FC (28.6%) were also in the Cow AI group.The smaller follicle coexisting with the cystic follicle may have matured and this follicle may have led to estrus.On the other hand, 29 cows (37.7%) were in the Cow PG group and the follicle coexisting with the cystic follicle may have ovulated and formed the CL in those cows.
There is another mechanism for recovering from cystic ovaries by GnRH treatment.Following GnRH treatment, luteinization of cysts occurs, which is followed by luteolysis and the cows resume a normal estrus cycle (Peter, 2004).Because the luteinization and luteolysis takes about 16 to 18 days (Peter, 2004), the rate of receiving AI within 16 days of GnRH administration in Group FC may have tended to be low.Furthermore, some cows in Group FC may have been judged as belonging to the Cow NE group, even if GnRH showed effectiveness.
A single GnRH treatment can induce ovulation of dominant follicles (Crowe et al., 1993;Quintans et al., 2004) or restart ovarian cyclic activity (Cavestany and Foote, 1985).In this study, 62 cows (37.3%) in Group FOQ and 12 cows (27.3%) in Group OQ were in the Cow AI group.GnRH may have stimulated the growth of dominant follicles, which resulted in estrus in those cows.The rate at which estrus was induced in Group FOQ tended to be higher than that in Group OQ.It was reported that the smaller follicles are more likely to become atretic follicles than follicles having a diameter of more than 8.5 mm (Roche et al., 1998) and the ovaries were considered to be active when follicles with a diameter greater than 8 mm are present (Sheldon et al., 2000), Furthermore, the larger the follicle which was induced ovulation by GnRH, the higher the conception rate was (Peters and Pursley, 2003).Therefore, GnRH is recommended as the treatment for cows with ovarian quiescence that has a follicle of which the diameter is more than around 8 mm.If not, other formulations which stimulate follicle growth, like eCG, can be used as a pretreatment to the administration of GnRH in order to enhance the effectiveness of GnRH.However, 52.3% of the cows were in the Cow AI or Cow PG groups, even in the absence of a follicle with a diameter of more than 8 mm.The conception rate for the Cow AI group was 40.6% and this rate was the as same as for cows in which the natural estrus was detected by the observation of estrus signs (Cavestany et al., 2007;Cordoba and Fricke, 2002;Cvestany et al., 2003;Tóth et al., 2006).Accordingly, the administration of GnRH at the reproductive checkup is recommended for cows with ovarian quiescence.
When cows experience reduced PG level before ovulation, short luteal phases caused by the premature release of PGF 2α by the endometrium may occur (Cerri et al., 2011).Ovulation may have progressed in the Cow AI group as follows: the first follicle with a diameter of more than 8 mm was released in the ovulation after GnRH administration, then the CL that developed following the ovulation regressed early and the cows showed estrus.Some cows in Group FOQ might have followed this pathway.The ratio of cows in the Cow PG group was almost that same in Groups FOQ (28.3%) and OQ (25.5%).Although dominant follicles are considered to be luteinized without ovulation, there is a possibility that the estrus signs were overlooked, even if estrus was induced and the rate of reaching the Cow AI group may be increased through more careful observation.
Pretreatment by intravaginal device or hCG aims to raise the blood progesterone level and expose follicles to sufficient progesterone level and the administration of GnRH after that pretreatment can effectively induce ovulation and maintain normal CL function (Mee et al., 1991;Stevenson et al., 2007;Smith et al., 1987).From these reports, pretreatment by intravaginal device or hCG may be valuable if GnRH is given to cows with ovarian cyst or quiescence but in the absence of CL.
This study shows that the application of ultrasonography improves the effectiveness of the reproductive checkups due to the visualization of the ovarian structure and, furthermore, the administration of GnRH in a one-time administration to cattle with ovarian cyst or quiescence but without CL at regular reproductive checkup may shorten the period to conception.

Table 1 :
Treatment and effect of GnRH administration