The non-surgical embryo collection has resulted in a widespread use of non-surgical techniques in bovine species because of greater research and subsequent development likewise, consequent progress in non-surgical embryo collection due to an increased number of studies in goats has allowed its application in the future. Researchers have paid particular emphasis on enabling comprehensive non-surgical methods in goats to recover embryos, given the growing concern and limitations regarding animal welfare (Fonseca et al., 2016). The sheep and goats have benefited from the use of assisted reproductive technology to enhance their genetic makeup and reproductive efficiency (Cognie et al., 2003; Talukdar et al., 2022). Assisted reproductive technologies (ARTs) offer planned reproduction to fulfill the livestock product demands of a growing population. Trans-cervical/non-surgical embryo recovery (NSER), was first successfully used in 1984 to harvest embryos non-invasively from dairy goats (Bondurant et al., 1984). The main advantage of NSER is the reduced risk or absence of adhesion, making multiple collections more feasible compared to laparoscopy, which had 70% adhesion in third recoveries, while laparotomy showed 100% adhesion in second recoveries (Andrioli et al., 1999). The trans-cervical technique for embryo recovery has challenges like placing the catheter into the uterus through cervix, inability to manipulate the reproductive tract rectally in goats. To address these challenges, the injection of oxytocin and prostaglandin has been used (Suyadi and Holtz, 2000). A 100% success rate in trans-cervical collection was achieved when prostaglandin was administered hours before embryo collection (Fonseca et al., 2022). A study on nulliparous goats investigated the use of oxytocin to induce cervical dilation for trans-cervical insemination when administered intravenously 10-15 minutes before artificial insemination, finding that oxytocin administration led to greater cervical dilation and increased catheter penetration depth (Gutierrez et al., 2022). The potential of oxytocin treatment to improve embryo collection efficiency, for instance, by optimizing dosage, should not be overlooked (Suyadi and Holtz, 2000). Although research into trans-cervical embryo collection continues, no practical technique is yet recommended for field use (Mobini, 2019). This study considers the importance of cervical dilation for uterine access in non-surgical embryo recovery in goats.

The study was conducted at the Department of Animal Reproduction, Gynaecology and Obstetrics and the Instructional Livestock Farm Complex (Goat Unit), College of Veterinary Sciences and Animal Husbandry, Central Agricultural University, Aizawl, Mizoram (23.73^oN, 92.71^oE; mean annual temperature 21.6^oC). Additional work was carried out at the Goat Breeding Farm, A.H. and Veterinary Farm Complex, Thenzawl, Mizoram (23.27^oN, 92.77^oE; mean temperature 21.3^oC). Twelve healthy, cyclic Beetal does (2-4 years; Average 30-35 kg; BCS 3.0-3.5) reared under a semi-intensive system were selected. All animals were vaccinated, dewormed and screened by abdominal palpation followed by ultrasonography. They were randomly divided into two equal groups (n = 6). Standard estrus sync-hronization, superovulation, natural mating and non-surgical transcervical embryo recovery procedures were applied.

For estrus synchronization and superovulation all does received intra-vaginal progesterone-impregnated sponges (Avikesil-S®,Division of Animal Physiology and Biochemistry, ICAR-CSWRI) for 7 days and at sponge withdrawal, 125 µg Cloprostenol sodium (Pragma, Intas Pharmaceuticals limited) was administered intramuscularly (Babu et al., 2020). Twenty-four hours prior to sponge removal, 800 IU PMSG (Folligon®, MSD Animal Health) was injected intramuscularly (Mohamad et al., 2014). At the time of first mating, 1000 IU hCG (Chorulon®, MSD Animal Health) was administered intravenously for ovulation synchronization (Nagashima et al., 1987). Estrus was monitored at 12-hour intervals or before after sponge removal. Estrus response (%), estrus induction interval (time from sponge removal to onset of estrus) and estrus duration (time from onset to end of standing estrus) were recorded individually. Does in estrus were naturally mated with fertile bucks.
 
Cervical dilatation protocols
 
Group I: 37.5 µg d-cloprostenol (Pragma-D®, Intas Pharmac-euticals limited) intramuscularly 12 h prior to embryo collection.
Group II: 37.5 µg d-cloprostenol (IM) 12 h prior + 50 IU oxytocin (Evatocin®, Neon Laboratories Limited) intravenously 20 min prior to embryo collection.
Donor Preparation and anaesthesia: The perineal region was cleaned and prepared aseptically. Epidural anaes-thesia was administered using 1-2 ml of 2% lignocaine (Lox 2®), Neon Laboratories Limited) at the sacro-coccygeal space.
Trans-cervical penetration procedure: A lubricated vaginal speculum was inserted and the cervical os was graspedwith Allis forceps under visible torch light and bring at the level of Vulva (Fig 1). A 16 FG silicone 2 way Foley catheter (Silko-Cath®, Romsons Group Private Limited) with a stainless-steel stylet was gently advanced through the cervical canal into the uterine body in rotatory manner by preventing cervical folds with maintaing stiffness of catheter with stylet.
Trans-cervical penetration success rate: Success was recorded based on complete catheter passage through the cervix, when resistance become loss and balloonbecome inflate.
  

 

 


Following successful catheterization and fixation of catheter ballon by pulling backward tightly against internal-OS of cervix, a commercial flushing medium (EUROFLUSH®,  IMV Technologies) was infused 15-20 ml in each flush total 120 ml into the uterus and recovered using a 50 ml catheter-tipped syringe. Infused and recovered volumes were measured for each doe.
 
 

 

 
The recovered medium was filtered using an EZ filter (IMV Technologies) to retain embryos. The filtrate was examined under a stereo zoom microscope and embryos were counted individually.
 

 

 
Embryos were evaluated and graded (Grade 1-4) according to international embryo eransfer society (IETS) criteria under Stereozoom microscope.
 

 

All the reproductive parameters were depicted in the Table 1. The oestrus response was 100% in both Group I and Group II. Oestrus was confirmed through behavioural and physiological signs in all synchronized does, indicating the overall effectiveness of the synchronization protocol used in this study. These findings are consistent with earlier reports by Palanisamy et al., (2015), who observed full oestrus response using CIDR-based synchronization and PMSG was administered for superovulation and hCG for inducing synchronized ovulation. In contrast, Sumeldon et al., (2015) reported a lower oestrus response of 70% in the HSM + GnRH group. Such variation might be attributed to differences in breed, age, synchronization protocols and overall management conditions and individual variation (Barik et al., 2025).



The mean±SE oestrus induction interval was 17.83±4.08 hours in Group I and 14.50±1.23 hours in Group II, with no statistically significant difference between the groups. This indicates that the onset of oestrus following sponge removal was comparable in both treatment groups. The induction interval recorded in Group I (17.83 hours) closely aligns with the findings of Palanisamy et al., (2015). It was shorter than that reported by Goel and Agarwal (2005), who documented an interval of 38.4 hours. This variation may be attributed to the dual FSH- and LH-like actions of PMSG, which stimulate follicular development and advance ovulation, resulting in earlier and more synchronized oestrus expression (Salleh et al., 2021). The accelerated preovulatory LH surge induced by PMSG likely contributed to the reduced interval observed in this study. Differences in the type of synchronization hormone may also explain the shorter induction interval. The current study utilized intravaginal progesterone devices, whereas Goel and Agarwal (2005) employed prostaglandin-based synchronization, which could account for the longer induction period reported in their study. Although PMSG dosages were similar-800 IU in the present work and 750 IU in the study by Goel and Agarwal (2005) the slightly higher eCG dose used here may have further contributed to the shorter interval, as eCG has been previously shown to reduce the time from sponge removal to oestrus onset.

The mean±SE oestrus duration was 44.16±3.38 hours in Group I and 46.83±3.17 hours in Group II, with no significant difference between the groups. The oestrus duration values recorded in this study are consistent with the findings of Palanisamy et al., (2015). Goel and Agarwal (2005) noted that higher estrogen concentrations associated with unovulated follicles can prolong oestrus duration, as reflected in their observation of longer oestrus in PMSG-treated compared to FSH-treated animals. Progesterone-based synchronization systems such as CIDR or intravaginal sponges suppress endogenous hormonal activity; following withdrawal, the rapid hormonal fluctuations induced by subsequent PMSG administration may lead to a more condensed oestrus period, particularly in shorter synchronization protocols.

The cervical penetration success rate in Group I was 100%, which can be attributed to the intramuscular administration of d-cloprostenol for cervical dilatation. This treatment enabled smooth trans-cervical passage of the Foley catheter for non-surgical embryo recovery, demonstrating the effectiveness of the cervical dilatation protocol. The ease of catheter insertion indicated adequate cervical relaxation under the present study conditions. These findings are similar with the reports by Fonseca et al., (2022). Comparable results were also reported by Suyadi et al., (2000), who achieved a 90% penetration rate in goats. Cervical relaxation in such treatments is primarily associated with intrinsic changes in the extracellular matrix, contributing more to cervical opening than smooth muscle relaxation (Fonseca et al., 2022).

The cervical penetration success rate in Group II was also 100%, achieved using a combination of intramuscular d-cloprostenol and intravenous oxytocin. This combination facilitated trans-cervical catheterization for non-surgical embryo recovery. Although effective, the combined protocol did not produce as pronounced cervical dilatation as d-cloprostenol alone. However, a slight increase in cervical canal width was observed in some does, as assessed using cervical dilators. These findings are consistent with previous reports by Pereira et al., (1998). The results exceed those of Lima-Verde et al. (2003), who reported lower penetration success using 50 µg cloprostenol administered 24 hours before embryo recovery, likely may due to differences in dosage and protocol.

Prostaglandins play a major role in cervical dilatation by softening the collagen matrix of the cervix. Although oxytocin was expected to enhance catheter passage when combined with cloprostenol, catheter insertion remained difficult in a few animals, likely due to Grade 2-3 cervices with pronounced cervical folds and a crooked canal (Kanthawat et al., 2024). Overall, both protocols proved effective in enabling successful trans-cervical catheterization for non-surgical embryo flushing in goats.

The average flushing media recovery rate in Group I was 41.94%, on day 7 of post-mating. This outcome was achieved using a single intramuscular dose of d-cloprostenol for cervical dilatation. The use of d-cloprostenol alone was not highly effective for non-surgical flushing media recovery. The finding was comparable to the 43.5% reported by Lee et al., (2015). The slightly lower recovery in the present study, despite similar uterine sites, media volume and flushing techniques, may be attributed to the use of a 16 Fr Foley catheter instead of the 8 Fr catheter used by Lee et al., (2015). Additionally, reduced media recovery may have resulted from excessive leakage due to high infusion pressure during flushing. The average flushing media recovery rate in Group II was 94.03%, on day 7. This high recovery rate was achieved using intramuscular d-cloprostenol administered 12 hours before flushing in combination with intravenous oxytocin given 20 minutes prior to the procedure, which likely enhanced uterine contractions and facilitated efficient flushing. The recovery rate in Group II (94.03%) was statistically higher than that of Group I (41.94%), demonstrating the superior effectiveness of the combined protocol. The improved flushing efficiency in Group II may be due to the rapid action of intravenous oxytocin, as Jain and Gautam (2024) reported that oxytocin administered intravenously acts almost immediately, achieving peak concentration within 30 minutes and inducing effective uterine contractions.

In Group I, the average number of embryos recovered per doe was 0.0 despite oestrus synchronization, superovulation and natural mating. These results indicate that d-cloprostenol alone was ineffective in retrieving embryos under the conditions of this study. The finding of zero embryos recovered is consistent with Lee et al., (2015), who reported no embryo recovery using non-surgical flushing with a similar catheter, uterine site and method, largely due to insufficient flushing from the uterus. These results highlight that achieving a higher flushing media recovery is critical for successful embryo collection.

In Group II, the average number of embryos recovered per doe was 0.67 following combined intramuscular d-cloprostenoland intravenous oxytocin administration for cervical dilatation. Oxytocin was administered slowly via IV to induce rhythmic uterine contractions, which likely facilitated embryo recovery. Although Group II achieved a high flushing media recovery rate but the number of embryos retrieved remained low and was not statistically significant compared to Group I. The low recovery contrasts with reports by Lee et al., (2015), who recovered an average of 4.5 embryos per doe with oxytocin and Morais et al., (2020), who recovered 3.6 embryos per doe without oxytocin. The likely reason is a weaker superovulatory response induced by PMSG, as suggested by Goel and Agarwal (2005), indicating that higher media recovery alone does not ensure successful embryo retrieval. Additional factors influencing low embryo recovery include the uterine site, as Nagashima et al., (1987) reported higher embryo recovery from the uterine horn between days 5-7 than from the uterine body used in the present study. Armstrong et al., (1983) noted that embryo recovery becomes difficult after day 5 when PMSG is used for superovulation. Breed-specific differences may also contribute, as Beetal goats exhibit lower embryo recovery rates compared to Assam Hill goats (Borah et al., 2012). High post-oestrus estrogen levels may further reduce embryonic development and recovery (Greve et al., 1995). Overall, while the combination of prostaglandin and oxytocin improved cervical passage and flushing efficiency, embryo recovery remained limited due to superovulatory and physiological factors.

In Group II, the morphological quality of recovered embryos was as follows: Grade 1= 75% (2Morula, 1 Blastocyst), Grade 2= 25% (1 Morula) and Grades 3 and 4-0.0%. The combination of intramuscular d-cloprostenol administered 12 hours before flushing and intravenous oxytocin for cervical dilatation facilitated successful trans-cervical passage of the catheter, resulting in the recovery of embryos with predominantly good morphological quality. Grade 1 embryos were significantly more frequent than Grade 2 embryos, indicating the effectiveness of the combined protocol in retrieving high-quality embryos.

Compared to previous studies, the proportion of Grade 1 embryos in the current research was higher than reported by Morais et al., (2020). The proportion of Grade 2 embryos was comparable with Morais et al., (2020) but lower than Lima-Verde et al., (2003). These differences may be attributed to variations in breed, treatment protocols and timing of embryo collection.

The lack of embryo recoveryin group I might be attributed to factors such as insufficient superovulatory response with PMSG compared to FSH (Goel and Agarwal, 2005), reduced flushing efficiency and potential leakage of flushing medium during high-pressure injection. These results indicate that prostaglandin alone was inadequate for effective embryo retrieval in goats. Overall, the results indicate that oxytocin combined with prostaglandin significantly improved total embryo recovery per group compared to prostaglandin alone, highlighting the importance of cervical dilatation and uterine contractility in successful non-surgical embryo collection.

Cloprostenol alone or in combination with oxytocin can cause cervical dilatation in all goats. Nevertheless, it significantly increased the amount of flushing medium and the rate of embryo recovery. Embryo quality was assessable only in the oxytocin group, with Grade I embryos predominating, indicating improved embryo viability compared to cloprostenol alone. Compared to surgical procedures, the non-surgical approach to embryo recovery is easier and takes less time. It allows embryo recovery without the use of ultrasonography to locate the inflated balloon, thereby providing a practical possibility of embryo recovery in goats. The technique may be adopted at field and commercial levels to collect embryos non-surgically from elite animals.

The authors express their sincere gratitude to the Director, Department of Animal Husbandry and Veterinary, Government of Mizoram, Dr. R. Zothanmawii, for granting permission and providing institutional support to undertake this research work. The authors also acknowledge the invaluable cooperation extended by Dr. Ngursailova, General Manager and Dr. George Martin Lalruattluanga, Manager, Govt. Goat Breeding Farm, Thenzawl, Mizoram whose guidance and logistical assistance greatly facilitated the smooth execution of the study.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.