Superovulation in cows synchronized with two different progesterone + oestradiol protocols

A total of 26 Brown Swiss cows were used to compare the synchronization and superovulatory response of follicle stimulating hormone treated cows that were synchronized with progesterone+oestradiol valerate or benzoate. Control cows (n=8) were superstimulated with follicle stimulating hormone using twice daily injections with decreasing doses from day 10-13 after determined reference oestrus. Cows in treatment groups were received either ear implant (n=9) containing norgestomet+oestradiol valerate or progesterone releasing intravaginal device (n=9) containing progesterone+oestradiol benzoate, at random stage of the oestrus cycle, for 9 days. Seven days after the implant and progesterone releasing intravaginal device insertion, follicle stimulating hormone was injected as described in the control group. There was no significant difference between the groups for superovulation responses. In conclusion, both protocols synchronized the oestrus cycle in follicle stimulating hormone treated cows and, any of the protocols evaluated in this study can be used as a pretreatment for superstimulation started on the seventh day of the implant or progesterone releasing intravaginal device insertion in Brown Swiss cows.


Introduction
Success of embryo transfer depends on stimulating follicular growth, ovulation of these developed follicles, fertilization of oocytes, collection and transfer of embryos, establishment of pregnancy and finally obtaining many calves (Armstrong 1993, Mikkola et al. 2005, Novotny et al. 2005).Superovulation response is very important for this aim and it is one of the most critical stages in embryo transfer protocols.For this respect, it is very important to know about the factors effecting superovulation response and elimination of the negative situations (Andrade et al. 2003).
There are many researches in which greater superovulatory responses were reported when superstimulatory treatments were initiated 8-12 days after reference oestrus (Lindsell et al. 1986, Rajamahendran & Calder 1993).Gonadotrophin treatment started on 8-12 days after oestrus is based on the knowledge of emergence of the second follicular wave in these days (Hasler 1992, Huhtinen et al. 1992, Bungartz & Niemann 1994, Sato et al. 2005).However, the day of emergence of the second follicular wave has been shown to differ between twoand three-wave cycles and individual animals (Sirois & Fortune 1988, Webb & Armstrong 1998).It is reported that, only 20 % of the oestrus cycle is available for initiating treatment at the time of follicular wave emergence.That is, 80 % of the oestrus cycle is not conductive to an optimal superovulatory response (Bó et al. 2004).Some new protocols have developed to obviate this problem by exogenous control of both luteal and follicular function (Gordon 2005).In recent years, progestagen/progesterone+oestradiol combinations have been used to control of follicle wave emergence.This application also offers the advantage of initiating superovulatory treatments at a time that is optimal for follicle recruitment, regardless of the stage of the oestrus cycle (Nasser et al. 1993, Baracaldo et al. 2000).These new protocols permit the initiation of superstimulatory treatments at a self-appointed time (Bó et al. 2006).However, the interval to emergence of a new follicular wave is quite variable in cows treated with different oestradiol preparations and, initiation day of gonadotrophin treatment following these various preparations has a great importance on superovulation response (Colazo et al. 2005).In a study, superstimulatory treatment in cows initiated 4 days after treatment with 2.5 mg oestradiol benzoate or 5 mg or 2.5 mg oestradiol-17β+progesterone resulted in superovulatory responses comparable to those initiated 8-12 days after reference oestrus (Mapletoft & Bó 2004).However, Mapletoft et al. (1999) reported less synchronous emergence of a follicular wave and a lower superovulatory response in cows superstimulated 5 days after treatment with oestradiol valerate and 3 mg norgestomet.In a previous study, synchronization of the oestrus cycle using ear implant containing progesterone+oestradiol valerate and gonadotrophin injection for 4 days started on day 7 of the progesterone+oestradiol treatment was successful and, comparable superovulatory results determined with the protocol in which superstimulation was initiated on day 10 after spontaneous reference oestrus (Bülbül et al. 2010).
Because of the probability of difference between oestradiol valerate and oestradiol benzoate in synchronizing oestrus, the objective of the present study was to compare the synchronization success and superovulatory response of gonadotrophin injection started on the seventh day of the progesterone+oestradiol valerate or progesterone+oestradiol benzoate as a pretreatment for superstimulation in Brown Swiss cows.

Animals
A total of 26 lactating Brown Swiss cows, aging 3-8 years were randomly allocated into three groups.The animals were selected taking the criterions listed as followed: a) no dystochia and retained fetal membranes in previous calving; b) no prulent discharge during vaginal examination; c) 50-120 days post-partum; d) have shown oestrus at least twice, and no artificial insemination or mating after previous calving.The cows were kept in similar conditions of handling and feeding and fed with a ration composed of corn silage, alfalfa hay and a concentrate-mineral mix, had ad libitum access to fresh water and housed in a free-stall confinement facility.

Experimental protocol
Experimental desing was illustrated in the Figure 1.In the Control group (n=8), cows were superovulated with follicle stimulating hormone (FSH) (Folltropin-V, total: 400 mg NIH-FSH-P1, Bioniche Animal Health Inc., Ontario, Canada), from day 10-13 (spontaneous reference oestrus=day 0), using twice daily injections with decreasing doses (4, 4, 3, 3, 2, 2, 1, 1 ml) and intramuscularly injected 150 µg of D-cloprostenol (Dalmazin, Vetaş, İstanbul, Turkey) with the fifth FSH injections.In the treatment groups, cows were received either ear implants (Implant group, n=9, Crestar, Intervet, İstanbul, TURKEY) containing 3 mg norgestomet+5 mg oestradiol valerate or progesterone releasing intravaginal device (PRID) (PRID group, n=9, PRID, Sanofi Doğu İlaç A.Ş., İstanbul, Turkey) containing 1.55 gr progesterone+10 mg oestradiol benzoate at random of the oestrus cycle.Ear implants were inserted to the ear subcutaneously and PRIDs were placed to the anterior vagina of each cow for 9 days.Cows in the Implant group were also treated intramuscularly with 3 mg norgestomet+5 mg oestradiol valerate at the time of implant insertion.On the seventh day of the implant and PRID insertion, FSH was injected for 4 days as described in the control group.D-cloprostenol at the dosage of 150 µg was injected to the cows in the treatment groups with the fifth FSH injections.
Observation of oestrus was initiated following the last FSH injection.The cows in all groups were observed for oestrus signs (visual observation of standing heat, vaginal discharge) three times a day for 30 min each time and, oestrus was confirmed by rectal palpation of fluctuant dominant follicles and uterine tonus.All cows were artificially inseminated twice by a skilled technician 12 h and 24 h after the onset of oestrus and, were inseminated using frozen-thaved semen (20 × 10 6 spermatozoa/straw with about 60 % progressive motility after thawing) throughout the experiment.
Superovulatory response was assessed by rectal palpation and ultrasonography (PIE Medical Falco 100; Esaote, Maastricht, The Netherlands) 7 days after the first insemination, with regard to the number of corpora lutea in the ovaries using a 7.5 MHz linear-array transducer.The ovaries were scanned in several planes to identify all corpora lutea.Embryo flushes were performed non-surgically, 7 days after the first insemination, using 1 000 ml ringer lactate (Ringesol, Vilsan, Ankara, Turkey) containing 1 % fetal calf serum (N-4267, Sigma-Aldrich, St. Louis, MI, USA) and 0.1 % kanamicyn sulfate (Kanovet, Vetaş, İstanbul, Turkey) with a two-way disposable Foley catheter, inserted into the uterine horn through the cervix and, the ova/embryos were collected.The recovered lavage fluid was filtered through 72 μm filter (Emcon filter, Minitüb GmbH, Tiefenbach, Germany).Quality of the recovered embryos was assigned under a stereomicroscope at 20-fold magnification according to the morphological criteria of quality and viability determined by International Embryo Transfer Society (Wright 1998).Embryos were classified as grade 1 (excellent or good), grade 2 (fair), grade 3 (poor) and degenerated embryos and unfertilized oocytes.Grade 1, 2 and 3 embryos were defined as transferable.

Statistical analyses
The Mann-Whitney nonparametric test was used to compare the mean numbers of corpora lutea, total ova and embryos, grade 1, 2 and 3, transferable and degenerated embryos and unfertilized ova between the groups.Last FSH injection-oestrus intervals were compared using analysis of variance (One-way ANOVA).All analyses were carried out using a statistical analysis system configured for computer (MINITAB rel.12.1, Minitab Inc., State College, PA, USA).The differences were considered significant at P<0.05.

Results
All cows used in this trial showed signs of oestrus within 24 h of the last FSH injection.Last FSH injection-oestrus intervals were similar in groups (Table 1).
Recovery rates, the mean numbers of corpora lutea, total ova and embryos, grade 1, 2 and 3, transferable and degenerated embryos and unfertilized ova in Control, Implant and PRID groups were summarized in Table 1.There was no significant difference between the groups in any of the criterions that were evaluated.In Implant group, the number of corpora lutea and total ova and embryos were higher whereas the number of transferable embryos was lower than the other groups.However, the differences among groups were not significant.

Discussion
Treatments using oestradiol and progesterone have been widely used over the past years in oestrus synchronization (Burke et al. 1999, Martinez et al. 2002).In these protocols, a progestogen/progesterone device is inserted for 7-12 days and oestradiol and progestogen/ progesterone is administered on day 0 to synchronize follicular wave emergence (Diskin et al. 2002).Prostaglandin is injected the day before or at the time of device removal to ensure luteolysis (Lemaster et al. 1999).There are so many reports that achieved successful synchronization results using this protocol (Cavalieri et al. 1997, Penny et al. 1997, Ryan et al. 1999, Bülbül & Ataman 2006).The majority of cattle are expected to exhibit oestrus 24-48 h after removal of implants or intravaginal devices (Penny et al. 1997, Lemaster et al. 1999).It is reported that this protocol can be used to synchronize oestrus cycle as a pretreatment for superstimulation started 4 days after progesterone+oestradiol treatment Mapletoft & Bó (2004).The results of a previous study (Bülbül et al. 2010) led us to compare the synchronization success and superovulation response of two different protocols using progesterone+oestradiol and, superstimulation started on the seventh day of the progesterone+oestradiol application with the conventional superovulation protocol which was initiated on day 10 after oestrus in FSH superstimulated cows.In the current study, all cows in groups showed the signs of oestrus within 24 h after the last FSH injection.Last FSH injection-oestrus intervals were not significantly different between groups.In addition to the oestrus synchronization success in treatment groups was similar to that in Control group, there was no significant difference between treatment groups in synchronization.
It is reported that various factors influenced the superovulatory response of cattle (Kafi & McGowan 1997).One of these factors is the ovarian status at the time of gonadotrophin treatment and the timing of treatment with respect to the oestrus cycle (Colazo et al. 2005).It has shown that gonadotrophin treatments must be initiated at the expected time of the follicular wave emergence for optimizing superovulatory response (Adams et al. 1994, Mapletoft et al. 2009).Because of the day of the second follicular wave varies among cows and between cycles with two and three follicular wave (Sirois & Fortune 1988), synchronization of the follicular wave emergence prior to initiation of superstimulatory treatments is suggested (Gordon 2005).Progesterone+oestradiol treatment is said to cause a new follicular wave emergence within 3-6 days (Bo et al. 1995, Caccia & Bó 1998, Mapletoft et al. 1999, Diskin et al. 2002).
Oestradiol not only has been used to induce luteolysis in progestin-based protocols, but also has been shown to suppress antral follicle growth by suppressing circulating concentrations of FSH.Following metabolism of oestradiol, FSH surges and a new follicular wave emerges (Price & Webb 1988, Garcia & Salaheddine 2001).It is reported that the interval to emergence of a new follicular wave is quite variable in cows treated with different oestradiol preparations (Burke et al. 1999, Colazo et al. 2005).For this reason, initiation day of gonadotrophin treatment following these various preparations has a great importance on superovulation response (Colazo et al. 2005).In previous studies, Mapletoft & Bó (2004) reported comparable superovulatory results using superstimulatory treatment 4 days after treatment with 2.5 mg oestradiol benzoate or 5 mg or 2.5 mg oestradiol-17β+progesterone to those initiated 8-12 days after estrus while Mapletoft et al. (1999) reported lower superovulatory response in cows superstimulated 5 days after treatment with 5 mg oestradiol valerate + 3 mg norgestomet.In an other study, Bülbül et al. (2010) reported similar superovulatory responses in cows superstimulated 7 days after an ear implant insertion containing 3 mg norgestomet + 5 mg oestradiol valerate with conventional protocol in which superstimulation is started on day 10 after oestrus.In this study, superstimulatory responses in treatment groups were similar to each other, and also similar with Control group.This result may be originated from a new follicular wave emergence in cows treated with implant and PRID in the same manner.Similar to our results, there are some other researchers who reported progesterone+oestradiol treatment caused a new follicular wave emergence for implant (Bo et al. 1995, Martinez et al. 2000) and PRID (Bridges et al. 1999, Cavalieri et al. 2003) application.
The most important aim of this study was to compare the embryo quality obtained from different synchronization protocols.There was no significant difference among groups for the numbers of transferable embryos.In addition, the numbers of grade 1, 2 and 3 embryos were also similar among groups.In the light of the data obtained for embryo quality, we can regard both protocols as successful for oestrus synchronization prior to superovulation.
Estradiol usage in cattle is still legal in most countries around the world and it is being used extensively for oestrus synchronization programs (Butler et al. 2012, Manes et al. 2012) while its application is not allowed in food producing animals in the European Union.
Because of this, the effects of it have to be investigated to improve our scientific knowledge and to compare methods including estradiol with alternatives (e.g.progesterone, GnRHprostaglandin, double-prostaglandin etc.).
In conclusion, both of the progesterone+oestradiol treatments synchronized the oestrus cycle, in FSH superstimulated cows, in this study.Superstimulation started 7 days after synchronization of follicular wave emergence by progesterone+oestradiol valerate or benzoate treatment has resulted in comparable superovulatory response with conventional protocol which is started on day 10 after reference oestrus.That is, any of the progesterone+oestradiol valerate or benzoate protocols can be used for synchronization prior to superstimulation according to the results of this study.Another advantage of the synchronization of the oestrus cycle with progesterone+oestradiol is elimination of need for detecting oestrus or ovulation and waiting 8-12 days after oestrus to initiate gonadotrophin treatments.Findings of this research suggested that, exogenous control of oestrus cycle by using progesterone+oestradiol valerate or benzoate combination as an ear implant or PRID for 9 days, and gonadotrophin injection over a 4 day period initiated on the seventh day of the implant or PRID application, may offer the advantage of initiating superstimulatory treatments at a time that is optimal for follicle recruitment in Brown Swiss cows.
Figure 1Experimental design

Table 1
Recovery rates, the mean last FSH injection-oestrus intervals, numbers of corpus luteum, total ova and embryos, transferable, grade 1, 2 and 3 and degenerated embryos, and unfertilized ova in control, implant and PRID groups (±SEM)