The experiment procedure was approved by the Institutional Animal Care and Use Committee at Mahasarakham University Thailand (0008/2552).

The study was conducted at a commercial goat farm during October until April of the following year in Prachuap Khiri Khan Province, Thailand (Latitude: 11° 49′ 15.53″ N, Longitude: 99° 47′ 2.76″ E). The average body weight and body condition score of the goats were 30±2.5 kg and 3.0 to 3.5, respectively. During the experiments, goats were housed indoors on straw bedding, fed with hay ad libitum and a commercial concentrate diet having 16% protein, with water always available.

Prior to the experiments all the animals were scanned by ultrasonography (HS-2000 Honda Electronics CO. LTD, Toyohashi, Aichi, Japan) with trans-rectal probe inducer to confirm the healthy reproductive tracts and their non-pregnant status. All the nannies were synchronized to a common day of oestrus using intra-vaginal Progesterone sponges containing 65 mg medroxyprogesterone (Sincro-gest, Laboratorios Ovejero, Leon, Spain) for 12 days. At the time of sponge removal, nannies were injected intramuscularly with 250 IU pregnant mare serum gonadotropin (PMSG; Intervet Thailand Ltd., Bangkok, Thailand). At 24 h and 48 h after the sponge removal, the present of oestrous signs were observed by presenting the Billy goat outside the female goats’s pen, each time for 2 h.

A total of 110 cross-bred Boer-goats (57 nulliparous and 53 multiparous) were randomly allocated to 2 groups. All the goats were inseminated each time with 0.25 mL F-T semen containing 150×10⁶ spermatozoa. The F-T semen used for all the goats was from the same production batch and the same Billy goat and had a post-thaw progressive motility of 45%. Goats in Group 1 (26 nulliparous and 34 multiparous) and Group 2 (31 nulliparous and 19 multiparous) were inseminated twice at 52 h and 56 h after the sponge removal using cervical AI with or without prior topical application of HA. Topical application of 40mg HA was accomplished at 48h after the sponge removal (group 1 only) as described by Perry et al [26].

A healthy 3 years old Boer Billy goat was used in this study. The fresh semen was collected using the artificial vagina. The collected fresh semen was placed in a 37°C water bath prior to evaluation. Sperm motility, progressive motility and viability were evaluated. The quality of fresh semen was evaluated in which sperm motility, progressive motility and sperm viability were 95%, 80%, and 90%, respectively. After the evaluation, sperm cryopreservation was performed. The seminal plasma was removed by centrifuged at 1,500×g for 10 minutes using Lactate ringer solution with the proportion of semen:Lactatate ringer solution of 1:19. After that the supernatant was discarded leaving the sperm platelet. Then a commercial extender consisting of 34% of One-step (Continental Plastic corp., Delavan, WI, USA), 46% of distill water and 20% of egg yolk was added gently onto the semen platelet at 37°C to make up the semen extender. The concentration of sperm extender was 600×10⁶ spermatozoa/mL. The semen extender was loaded into 0.25 mL straws which made 150×10⁶ spermatozoa per straw. The semen straws were gradually cooled down in 4°C cabinet for 4 hours. After that all semen straws were frozen at 4 cm above the liquid nitrogen level for 13 minutes then were stored in the liquid nitrogen at −196°C. Twenty four hours following the liquid Nitrogen storage, sperm quality was evaluated after thawing. The frozen semen was thawed by placing the frozen semen straw into the 37°C water bath for 30 seconds, then the thawed semen was dropped on 37°C warm glass slide and the sperm motility, progressive motility and viability were evaluated at 400 magnification under phase contrast microscope. In the present study, the F-T semen with sperm motility, progressive motility and sperm viability of 69%, 70%, and 52%, respectively was used for AI.

The rear part of the goat was placed over a rail. The rear end of the goat was firmly and gently restrained by the inseminator’s assistant. The vulva and the surrounding area were cleaned using a disinfectant wipe and let air dried prior to the insertion of the duckbill vaginal speculum which was fitted with a light source to locate the site of the cervical opening. Once the cervical opening was located, cervical artificial insemination (CAI) was performed using the duckbill vaginal speculum lubricated with the non-toxic gel for spermatozoa. The F-T was thawed by placing the straw into the 37°C water bath for 30 seconds after that both ends of the straw were cut letting the F-T semen run into the 1.5 mL tube which placed in the 37°C heated block. The amount of 0.25 mL F-T semen was loaded into the inseminating pipettes which attached with 1 mL syringe. The F-T semen was deposited using an inseminating pipette which was a modified 6F cardiac catheter (diameter = 2.0 mm), 35 cm long, brite tip sheath introducer (Cordis, Johnson and Johnson company, Miami Lakes, FL, USA) attached to a 1 mL tuberculin syringe. The cervical opening was located and then the tip of the inseminating pipette was inserted into the cervical canal with the help of a stylet and was gently pushed through the cervical canal without any force so as to achieve maximum penetrability. Once at place, stylet was removed, and F-T semen was deposited by pushing the plunger of the 1 mL syringe.

The site of semen deposition was recorded for each ani mal. When the F-T semen was deposited at the opening of the cervix the “cervical os” was recorded, or “intra-cervix” was recorded when the inseminating pipette was passed into the cervical canal but no further, then the inseminator deposited the F-T semen in the cervical canal. Lastly the “intra-uterus” was recorded when the inseminating pipette was passed through the cervical canal reaching into the uterine body where the F-T semen was deposited.

All the goats were scanned to diagnose pregnancy 42 days after the insemination using ultrasonography scanner (HS-2000; Honda Electronics CO., LTD., Japan) with trans-rectal probe inducer (7.5 MHz). The presence of the foetus and foetal membranes ratified the pregnancy.

The pregnancy rates or the site of semen deposition were calculated and compared between the 2 treatment groups using chi-square statistical analysis. The chi-square statistic calculated from the 2×2 crosstab table testing the independence between the two classifications. The statistical package used in this study was SAS University Edition 2018 (SAS Studio Release: 3.8 [Basic Edition] SAS Institute Inc., Cary, NC, USA). The differences between the groups were considered significant at 5% probability (p<0.05).

The overall pregnancy rate in the goats inseminated with F-T semen was significantly higher (p<0.004) with (63.3%) than without (36.0%) topical application of the cervix with HA (Table 1).

In nulliparous goats, pregnancy rate was significantly higher (p<0.045) with (65.4%) than without (38.7%) topical application of the cervix with HA. The same was true for multiparous goats and the pregnancy rate was significantly higher with (61.8%) than without (31.6%) prior cervical application of HA, respectively (Table 1). There were no differences in the pregnancy rates between the nulliparous and multiparous goats when inseminated with or without topical application of the cervix with HA (Table 1).

Overall, the topical application of the cervix with HA significantly increased the number/percentage of goats in which semen could be deposited deeper within the reproductive tract. At first insemination (52 h after the sponge removal), regardless of parity, in Group 1, percentage of goats with semen deposited at cervical os, intra-cervix and intra-uterus was 56.7%, 23. 3%, and 20.0%, respectively (Table 2).

At the time of second insemination (56 h after the sponge removal) illustrated that regardless of parity, in group 1 (with cervical topical application of HA) 50% of goats were inseminated at the cervical os, 30% at intra cervix, and 20% were inseminated intra-uterus.

Without topical application of the HA (group 2), at both first and second insemination, semen could be deposited at the cervical os only and none of the goats in this group had semen deposited at intra cervix or intra uterus (Tables 2, 3).

In nulliparous goats, with cervical topical application of HA, both at first and second insemination (52 h and 56 h after sponge removal, respectively), semen was deposited in 69.2% of goats at the os cervix and in 30.77% of goats at the intra-cervix. HA application significantly (p≤0.05) increased the percentage of nulliparous goats in which semen could be deposited intra-cervix compared to those without HA (group 2). However, even with HA application, in none of the nulliparous goats semen could be deposited into the uterus (Tables 2, 3). In multiparous goats, HA application, not only significantly (p≤0.05) increased the percentage of goats that were inseminated intra-cervix but also allowed 35% of animals to be inseminated intra-uterus. In both nulli- and multi-parous goats, at both inseminations, without HA application, semen could only be deposited at the os-cervix and none of the goats were inseminated intra-cervix or intra-uterus (Tables 2, 3).