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8  

Effect of the administration of one FSH single dose in superovulation treatment in Corriedale Booroola ewe�s embryos production.


D. Fern�ndez-Abella1,2*,  I. Lago2, M. Rodr�guez1,2, O. Irabuena1,3, S Sterla1,3. 


 

1. Polo de Desarrollo Universitario en Producci�n y Reproducci�n en Rumiantes. UELAR. Regional Norte. Salto. Uruguay.

 2. Secretariado Uruguayo de la Lana, Rbla Baltasar Brum 3764, Montevideo 11800, Uruguay. 

3. Laboratorio de Inmunolog�a. UDELAR. Regional Norte. Salto. Uruguay.

*Corresponding author: Daniel Fernandez Abella. E-mail: HYPERLINK "mailto:ferabe@unorte.edu.uy"ferabe@unorte.edu.uy





Introduction 
      Embryos production by means of multi-ovulation and embryos transfer (MOET) contributes to the genetic improvement in ewes, because it does not require the use of elite rams and to produce embryos from the best females. At these days, MOET Programs based in a twelve to fourteen days exposure to progesterone, including FSH in order to obtain superovulation are used [1-3]. The exposure to progesterone can be reduced by using the first follicular wave [4]. Anyway, the treatments including FSH start two days before the vaginal pessaries are retired. Usually, six to eight doses by day are applied [5,6]. Other hormones (eCG y GnRH) are used which determine a repeated animal manipulation, with the consequent increase in the labour costs. 
Consecutive FSH treatments lead to reduce the response, especially after the third treatment, independent of the time of the year [6, 7]. This indicates that every FSH reduction level might allow the adverse effects of the succeeding treatments in case that those treatments are effective.   
In cattle, through the last fifty years, several researchers have progressed in simplifying superovulation protocols [8-11]. Nowadays, treatments allow superstimulation with a single FSH treatment or two treatments every 48 hours, reducing the need to handle animals during gonadotrophin treatments.
On the other hand, there is very little information about treatments of embryos production with a single dose in small ruminants. Batt et al. [12]  reported the use of a single FHS dose in goats. Rutigliano et al. [13-14]  used a single dose of recombinant follicle stimulating hormone agonist on the superovulatory response of sheep.
The objective of this research was to quantify the reduction of the FSH treatment to a single dose, comparing its administration at different moments regarding the progesterone vaginal pessaries removal.     
Materials and  methods
Animals and treatments
The research work was located at the Investigation and Research Centre of the Uruguayan Wool Secretariat (SUL, in Spanish) in Florida, Uruguay (33�52� S, 55�34� W). 
It included eighty-four females and five males Corriedale Booroola breed. The females were heterozygotes for the gen �Booroola� and the males were homozygotes.   
The ewes were multiparous, between three and five years old. They were selected from a group of eighty females, taking into account their backgrounds in terms of corpus luteum (CL) observed by means of laparoscopy (CL: 2-4) in a previous ovulation occurred before the treatments application.  The ewes had an average weight of 53.1 + 0.8 Kg and an average corporal female condition of 3 + 0.3. 
The multiovulation treatments were done at the beginning of the Southern Hemisphere winter (June 22). Seven treatments were evaluated combining different doses and timing of i.m FSH administration (NIH-FSH-P1; Folltropin�; Bioniche, Belleville, Canada) equivalent to 28 mg Armour units (400 mg NIH-FSH-P1).  
The synchronization protocol was the same for all the treatments. It consisted in: fourteen days of intravaginal pessaries with 60 mg of medroxiprogesterone (Progespon�,Synthex, Argentina), and the administration of  200 IU de eCG (Inducel 5000; Universal Lab, Uruguay) by the time when the pessaries were removed,  followed by the administration of  GnRH 35 h after (8.2 �g Buserelina acetate, Gonaxal, Biogenesis-Bago, Argentina).  For each protocol, the �zero day� was the one in which the vaginal sponges were placed. The substitution of the pessaries by new ones was done in the morning of the day nine and they were removed definitively on day fourteen. 
The treatments T1 (Traditional protocol used in Uruguay), T2 and T3 involved the administration of decreasing FSH doses 156 mg, 124 mg and 46 mg (four decreasing doses applied each one of them, twice a day, with a 12 hours interval). The treatments involving a single FSH dose were variable in terms of the application moment: 36, 24 and 12 hours before and simultaneously to the pessaries removal (T4, T5, T6 and T7 respectively).
2.2. Artificial insemination and embryo recovery
The ewes insemination was done using fresh sperm (five ram�s pool), in two ways: cervical (60 million sperms) and intrauterine laparoscopic (20 million sperms) forty and fifty five hours (respectively) after the vaginal sponges removal. 
The recovery was realized when the embryos development was between morulae and blastocyst, about the sixth or seventh day.
Using semi-laparoscopy technique, a posteriori of the ovulation rate observation, the uterine horns were exteriorized by doing a little incision (3 to 4 cm) done in the middle line. The horns washing or flushing (in antegrade way) was done from the utero-tubal junction to the basal part of the horn, following the technique described by Smith and Murphy [15], using modified phosphate-buffered saline (PBS). 
After the recovery, the ova were classified depending on two criteria:
Their phase of development : immature forms or non-fertilized, two to four cells, morulae, blastocyst; and
Their morphological appearance: excellent (category 1), good (category 2), poor (category 3), dead or degenerate (category 4), according to the IETS (International Embryo Transfer Society) classification [16].  
2.3. Statistical analysis
The statistical analysis was done using the GENMOD procedure SAS (SAS/STAT version 9.1, SAS Institute Inc., Cary, NC). The statistical significance level was determined in 5 %. 
Results 
Regarding to ovulatory rate, statistical significance difference (P<0.05) was obtained between treatments T1 and T2 compared with T3; the values were 10.9 y 12.5 versus 5.0; respectively (Table 2). Treatments T5, T6 and T7 did not show statistical significance difference regarding to corpus luteum, being the obtained values 8.9, 6.3 and 8.3 respectively. However, the obtained values for those treatments were lower than the ones obtained for T1 and T2. T4 turns to be non-viable because only three ewes did ovulate, showing only one corpus luteum. 
In terms of embryos production (Table 3), no statistical significance difference (P>0.05) was detected within those treatment that produced embryos (T5, T6 y T7) concerning to the amount of embryos obtained either by recovered ewe nor total ewes. Despite this, T6 was the most effective treatment in embryos production (number by collected ewe). However, considering the total ewes under treatment, T5 seems to be the best when considering the number of collected animals (it gave the higher number). Once counting the number of frozen embryos (by phase and corresponding quality) relative to the observed corpus luteum before the collect, T6 demonstrates higher efficiency when compared with T5 and T7. 
	A high percentage of the embryos were able to be collected and freezable (82.7% in those treatments with FSH decreasing doses and 75.2% in the ones with single FSH dose). This means that both quality and embryos development obtained were adequate, having morulae and blastocysts with excellent and good qualities (categories 1 and 2). T2 shown a higher number of embryos production (5.4 embryos by donor), followed by T6 and T2 (4.1; 4.0 embryos by donor, respectively), and T7 presented the lower efficiency in embryos production.
In terms of collected and freezable embryos number and quality, results showed that they had very good quality in general (categories 1 and 2 according to IETS) except by one (morulae) coming from T1, which showed poor quality (category 3) after the evaluation.
Discussion
When comparing the obtained ovulation rate with the values reported in the literature using doses of 100 mg de FSH (>7 mg Armour units) or higher, the results in Corriedale ewes were similar to those reported by Bonino et al. [17], 8.0 and 8.6 LC.  In Merino ewes results agreed with those reported by Jabbour and Evans [18], 11.1 and 11.4 LC. For Manchega breed Gonz�lez Bulnes et al. [2,19,20] obtained, depending on the year and case, 11.4; 11.1; 12.2 and 10.5 LC respectively. In Pelibuey breed, Ak� L�pez et al. [21] reported 12.4 and 8.2 LC, and in Merino breed, Menchaca et al. [9] obtained results of 10.3 and 13.5 LC. 
The possibility of reducing the FSH dose to values closer to 100 mg allows diminishing costs for each collected transferable embryo; good results were reported when using an 80 mg dose [6].
No antecedents were found for this treatment in ewes having the Booroola gene. Nonetheless, it is possible to endorse that it was a response to the treatments, because the ovulatory rates were superior to those observed in the same flock without treatment: 4.3-5.0 LC [22]. 
When evaluating the effects of a decreasing FSH dose application, results agree with the research done by Jabbour and Evans [18] and Batt [12]  whom reported that efficiency increases in terms of ovulatory rate when combining decreasing FSH dose and a single eCG application (due its longer middle life). This combination allows having a lower perturbation in both ovulatory levels and patterns of oestrogen and LH. At the same time, it lets create a more advantageous fertilization environment as well as an early embryo development inside the female reproductive tract. If oestrogen secretion is interrupted in any phase, it would provoke follicular atresia, which is undesirable for obtaining an adequate multiovulation [23].  
The GnRH use improves the response to the superovulatory treatment, by stimulating the release of the preovulatoire LH discharge [24]. GnRH administration 35 hours after the vaginal pessaries removal means the matching of a sudden LH discharge and a preovulatoire LH peak provoked by the high oestrogen concentration due to the collected and selected follicles (the ones with higher size); that stimulates the hypothalamus to release GnRH in a natural way. Eppleston et al. [25] verified, by means of its application 36 h and 44 h after the vaginal pessaries removal, a fast LH increasing 4 hours after the treatment. The same author concluded that, the ovulation time is anticipated by applying eCG 24 hours before the pessaries removal; also reported that the GnRH use makes shorter the period in which ovulation takes place. Wolff et al. [27], by the administration of one GnRh equivalent 36 h after the vaginal pessaries removal, obtained a fertilization rate between 70% and 80 % when the artificial insemination was done either 42 h or 55 h after the sponge�s extraction. In spite that in others researches done before that hormone was used to both guarantee and synchronize ovulation, other authors like Eppleston et.al, [25], Batt et al. [12] and Menchaca et al. [5] used a similar dose than that used in this research.
No bibliographical references were found about the use of one single FSH dose administration in ewes. The most similar reference is a research conducted by Batt et al. [12] in goats evaluating the response to treatments consisting in the use of two different FSH types �with different origin- and applying a single FSH dose combined with eCG, obtaining a variable number of corpus luteus (CL).
The literature does not report results applying treatments consisting in a single FSH concentrate dose in ewes. Rutigliano et al. [13,14]  using only a single injection analog of human FSH as an alternative to conventional gonadotrophin, that achieves a superovulatory response. On the other hand, as said before the research done by Batt et al. [12] in goats, evaluated the response to treatments using two different FSH types of different origin (from pig and sheep) applying a single FSH dose combined with eCG, obtaining a variable number of embryos and CL. Among the treatments applied by Batt et al. [12] the more similar results to the treatments evaluated in this research are those combining one single FSH dose: 12 mg of pig�s FSH applied 48 hours before the vaginal pessaries removal, with 200 IU or 400 IU of eCG at the same time. The ovulatory rates obtained were 8.7 and 9.9 respectively.
T3 treatment (low FSH dose), even giving evidence of ovarian activity, was not effective because the ovulatory rates were similar to those registered in the flock [22]. That agrees with the facts informed by Evans et al. [26](2001), working with Suffolk crosses, obtaining similar ovulatory rates values (2.5 � 0,3 and 2.0 � 0,3) varying the number of days with progestogens although using a 500 UI eCG dose. In other research work, Armstrong and Evans [1] applied the treatment using a dose of 1500-200 IU obtaining higher ovulatory rates: 7.7 � 1.4 and 3.5 � 1.6 respectively, as well as higher embryo collection, for Suffolk breed.   
In Merino breed, using high eCG doses, Jabbour y Evans [18] applied a single eCG dose (1200 IU) 48 hours before the vaginal pessaries removal reporting a number of 11.4 � 1.8 luteus corps (LC) average per animal. Therefore, the administration of a similar dose that the one used in that research or higher, anticipating the application moment, might improve the ovulatory response. This fact should be confirmed in future research studies.
In superovulation treatments, a variable number of ewes did not show response (ovulatory rates < 3). In this research, 100% of the ewes did respond. This is explained by the previous selection of the ewes to be tested and also, in part, because all of them had the prolific gene FecB. 
The most efficient superovulation treatments in both ovulatory rate terms, as in number and quality of collected and frozen embryos, are those in which a decreasing FSH dose is used. In the second place perform the treatments involving a single FSH dose administrated 24 h, 12 h or at the same moment when vaginal pessaries are retired.  In general terms, it can be stated that ewes having the Booroola gen behave as non-prolific animals when considering ovulatory response. Although no statistical significance difference was found between the treatments, T2 showed a high embryos production, followed by T6; the less efficient treatment was T7. Preliminary results in Corriedale non-prolific ewes (Fernandez Abella et al, unpublished) indicate the feasibility to administrate a single FSH dose 12 hours before the vaginal pessaries removal. In cattle, a single intramuscular injection of porcine FSH (200 mg) in 0.5% hyaluronan, permits superstimulation and embryo production in similar way consecutive FSH treatments do [28].
Regarding to the produced embryos they had a good quality and it was      possible to collect and freeze a great proportion of them, in the correct stage of development.
The Traditional Protocol (T1) and T2 used in non-prolific ewes resulted adequate for the prolific Booroola too. This research results shown that it is possible to produce transferable embryos with a single FSH dose (T6).
Acknowledgments
This work was financially supported by the Uruguayan Wool Secretariat (SUL).
Table 1.  Dose hormonal of treatments.
Table 2. Effect of multiovulation treatments on ovulation rate.   
Table 3. Effect of treatments on embryo recovery.
References
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