All experimental procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Khon Kaen University, Thailand (Approval No. IACUC-KKU-75/66). The ethical approval was granted for the period from November 2024 to June 2025 and covered all animal handling, hormonal treatments, AI procedures, pregnancy diagnosis, and follow-up monitoring conducted during this field study.

The study was designed and implemented in strict accordance with the institutional guidelines for the care and use of animals for scientific purposes and complied with internationally accepted principles of animal welfare. All procedures adhered to the Animal Research: Reporting of In Vivo Experiments 2.0 guidelines to ensure transparent and responsible reporting of animal research. Particular attention was given to minimizing animal stress, pain, and discomfort throughout the study.

Estrus synchronization, semen collection, and AI were performed by trained veterinarians using standard veterinary practices to ensure animal safety and welfare. Restraint during insemination was limited to the minimum duration necessary, and no surgical interventions or general anesthesia were employed. Animals were closely monitored before, during, and after all procedures for any signs of distress, injury, or adverse reactions. Post insemination monitoring was conducted for at least 48 h, and no severe complications attributable to the procedures were observed.

Pregnancy diagnosis by ultrasonography was performed using non-invasive techniques by an experienced veterinarian to reduce handling time and avoid unnecessary stress. All animals were maintained under normal farm management conditions with unrestricted access to feed and water throughout the study period. Any animal showing signs of illness or compromised welfare was to be withdrawn from the study and provided with appropriate veterinary care, although no such cases occurred.

Overall, the experimental design prioritized animal welfare while ensuring scientific validity, and all procedures were conducted in full compliance with ethical standards for animal research.

This field study was conducted between November 2024 and June 2025 on two commercial goat farms located in northeastern Thailand: Khon Kaen province (approximately 16.43° N, 102.83° E) and Sisaket province (approximately 15.12° N, 104.32° E). Both farms were officially certified as free from brucellosis and foot-and-mouth disease by the Department of Livestock Development, Thailand. The study areas are characterized by a tropical monsoon climate, with average ambient temperatures ranging from 26°C to 34°C and relative humidity between 60% and 75% during the study period. A short period of extreme heat was recorded in April, during which maximum temperatures reached approximately 36°C for 5–6 consecutive days.

A total of 37 multiparous native–Boer crossbred does aged 2–5 years, weighing approximately 30–50 kg and having a body condition score (BCS) of 3 on a 5-point scale, were enrolled in the study. All does were 3–4 months postpartum and met the predefined inclusion criteria. Animals with a history of reproductive disorders, current illness, or poor body condition (BCS < 2.5) were excluded.

The experiment followed a completely randomized design. Within each farm, does were randomly allocated to one of two insemination methods, C-TCAI or VE-TCAI, using a random number generator (Figure 1). At Farm 1, nine does were assigned to the C-TCAI group and eight to the VE-TCAI group. At Farm 2, nine does underwent C-TCAI and 11 underwent VE-TCAI; unequal group sizes resulted from the limited number of eligible animals available for allocation. All animals were managed under similar conditions across farms, with free access to dry grass and water. The same veterinarian performed all inseminations and pregnancy diagnoses to minimize operator-related variation.

Estrus synchronization was achieved using an intravaginal silicone progesterone-releasing device containing 0.3 g progesterone (CIDR®; Zoetis, USA) inserted for 13 days. On day 12, each doe received an intramuscular injection of 250 IU equine chorionic gonadotropin (eCG, Folligon®, Intervet, Germany) and 125 μg cloprostenol sodium (Estrumate®, Intervet/MSD, Germany) using 22G needles. Fixed-time AI was performed 48 h after CIDR removal. Two CIDR devices were lost during treatment, and the corresponding animals were excluded. No abnormal vaginal discharge or adverse effects were observed. Although formal estrus scoring was not applied due to the fixed-time AI protocol, behavioral signs of estrus, including vulvar swelling and clear mucus, were monitored, with approximately 20 does exhibiting visible mucus at the time of insemination.

Semen was obtained from a healthy, proven fertile 4-year-old Boer buck using an artificial vagina and immediately placed in a 35°C water bath. Semen processing, evaluation, and cryopreservation followed standard protocols with minor modifications [2]. Frozen semen straws were thawed at 37°C for 30 s prior to insemination. Progressive motility was assessed at 400× magnification using a phase-contrast microscope (Olympus, Japan), and only semen batches with post-thaw progressive motility exceeding 50% were used, as this threshold is associated with acceptable fertility in field AI programs for goats [2, 9].

All inseminations were performed by the same experienced operator using a single 0.25 mL semen straw containing 200 million spermatozoa to ensure procedural consistency. Prior to insemination, the vulva was cleaned and all instruments were lubricated with sterile gel. Does were restrained with their hindquarters elevated on a platform, with two assistants providing cranial and hindquarter support.

For the C-TCAI method, a 17-cm vaginal speculum was used to visualize the cervix with the aid of a penlight (Figure 2a), and semen was deposited using a 25-cm goat AI gun once cervical passage was achieved. For the VE-TCAI method, a Goat Visual Insemination Gun equipped with a 22-cm probe (1-cm diameter) and a 3.5-inch LED display (resolution 640 × 480 pixels) was used (Figure 2b). A 30-cm bovine AI gun was employed for semen deposition under real-time visual guidance, and semen was released only after clear visualization of cervical canal penetration. The actual applications of the C-TCAI and VE-TCAI techniques are shown in Figures 2c and 2d, respectively, while the endoscopic view of the goat cervix obtained using the VE-TCAI system is shown in Figure 2e.

Insemination time was recorded from insertion of the instrument into the vulva to complete withdrawal and categorized as ≤1 min or >1 min. The 1-min threshold was selected as a practical benchmark based on previous evidence linking shorter insemination times with reduced cervical manipulation, lower stress, and decreased semen backflow [19, 20]. After insemination, does were maintained with elevated hindquarters for approximately 2 min to facilitate semen movement into the uterus. Instruments were cleaned, rinsed with normal saline, and disinfected in 70% isopropyl alcohol for at least 2 min between animals [21]. All does were monitored for 48 h postinsemination for adverse reactions, with none observed.

Pregnancy diagnosis was conducted 45 days after insemination using transrectal ultrasonography with a Honda HS-1600V scanner (Honda Electronics Co., Japan) equipped with a 7.5 MHz linear probe. All examinations were performed by a single trained veterinarian to minimize inter-observer variability. Pregnancy was confirmed by the detection of an embryonic vesicle and fetal heartbeat. Pregnant does were subsequently monitored until parturition to record kidding rate, litter size, and neonatal outcomes.

Statistical analyses were performed using the Statistical Package for the Social Sciences version 29.0.2.0 (IBM Corp., Armonk, NY, USA) [22]. Fisher’s exact test was applied to compare insemination time categories (≤1 min vs >1 min) and pregnancy rates between insemination methods and farms. Statistical significance was set at p < 0.05.

Given the limited sample size, categorical analyses were prioritized, and effect sizes with 95% confidence intervals were calculated where applicable. The farm was treated as a random effect to account for potential environmental variation. The primary outcome measure was pregnancy status at 45 days post insemination, while secondary outcomes included insemination time, kidding rate, litter size, and adverse events. Although advanced regression modeling was not feasible, odds ratios and confidence intervals were calculated to provide quantitative estimates. Future larger-scale studies are planned to use mixed-effects logistic regression models incorporating relevant covariates, including parity, BCS, age, and post-thaw semen motility.

As summarized in Table 1, VE-TCAI resulted in a higher proportion of does being inseminated within 1 min compared with C-TCAI at both study sites. At Farm 1, 75.0% of does in the VE-TCAI group were inseminated within 1 min, whereas only 44.4% of does in the C-TCAI group achieved this benchmark. A similar pattern was observed at Farm 2, where 81.8% of does subjected to VE-TCAI required ≤1 min for insemination, compared with 33.3% in the C-TCAI group. When data from both farms were combined, 22 of 37 does (59.5%) were inseminated within 1 min, while the remaining 15 does (40.5%) required more than 1 min to complete the procedure.

Pregnancy outcomes following the two insemination methods are presented in Table 2. At Farm 1, pregnancy rates were comparable between the C-TCAI (33.3%) and VE-TCAI (37.5%) groups. In contrast, Farm 2 exhibited a higher pregnancy rate in the VE-TCAI group (45.5%) than in the C-TCAI group (33.3%). Overall, 14 of the 37 inseminated does (37.8%) were confirmed pregnant, resulting in the birth of 19 kids. When results from both farms were pooled, no statistically significant difference in pregnancy rate was detected between the two AI methods. The overall kidding rate was 51.4% (19 kids from 37 inseminated does), with an average litter size of 1.36 kids per pregnant doe. Twinning rates did not differ noticeably between the treatment groups. One abortion and one maternal death were recorded in the VE-TCAI group at Farm 2 during late gestation, while no stillbirths or neonatal mortality were observed in either group.

Both insemination techniques were generally well tolerated under field conditions. Minor vaginal bleeding was observed in one doe from the C-TCAI group immediately after insemination; this resolved spontaneously within a few hours without intervention. No cases of cervical trauma, infection, or other procedure-related complications were detected in any of the animals across both treatment groups.

This study evaluated the comparative efficiency of C-TCAI and VE-TCAI in goats under field conditions, with a particular focus on insemination time and pregnancy outcomes. The results clearly demonstrated that VE-TCAI significantly reduced the time required for semen deposition, with 78% of does inseminated within 1 min. Statistical analysis using Fisher’s exact test confirmed the significance of this reduction (p = 0.020), supporting the hypothesis that real-time visual guidance facilitates more efficient cervical navigation. These findings are consistent with previous reports in small ruminants, as well as in cats and dogs, where endoscopic assistance improved insemination accuracy and procedural efficiency [12–15]. Shorter insemination times are advantageous under field conditions, as they reduce handling stress and may enhance fertility. In nulliparous dairy goats, the highest pregnancy rates have been observed when semen deposition occurs within 20 s [19]. From a biological perspective, reduced insemination time limits cervical manipulation and irritation, decreases semen backflow, and minimizes stress-induced uterine contractions, thereby promoting deeper and more precise semen deposition and improving the likelihood of conception [19, 20].

In addition to improving procedural efficiency, VE-TCAI resulted in numerically higher pregnancy rates compared with C-TCAI, with an overall conception rate of 37.8% and a notably higher rate in Farm 2 (45.5%). These outcomes align with previous studies in dairy goat breeds, where visual-guided cervical insemination achieved pregnancy rates of up to 83% when semen was deposited in the hindquarters position using a 200 μL semen volume [9], and fertility rates of 55.9% when semen was placed near the uterine body [15]. Although the overall pregnancy rate in the present field study was lower, the consistent numerical advantage observed with VE-TCAI supports the role of visual guidance in improving insemination efficiency across different goat breeds and management systems under practical farm conditions.

The present findings reinforce previous evidence that endoscopic-assisted techniques can improve AI success under variable farm conditions. VE-TCAI represents a promising non-surgical alternative to LAI, which, despite its high effectiveness, requires anesthesia, surgical facilities, and specialized expertise, thereby limiting its routine use in field operations [16, 17, 19, 23]. By shortening procedural time and achieving comparable reproductive outcomes, VE-TCAI offers a practical alternative to both LAI and conventional TCAI, which is often constrained by anatomical and technical limitations. In preliminary field trials, attempts to apply deep cervical traction similar to the Embrapa technique caused noticeable discomfort in some does, leading to the preference for VE-TCAI as a minimally invasive approach that provides direct visual guidance without cervical traction. Other reported strategies to facilitate transcervical AI include the use of smaller insemination pipettes or chemical agents to induce cervical dilation [7, 18, 24]. Additionally, the size and length of the vaginal speculum may influence the success of conventional TCAI, as inadequate dimensions can hinder cervical visualization, whereas oversized instruments may cause discomfort or trauma, ultimately affecting semen deposition accuracy [25].

The procedural simplicity of VE-TCAI, combined with enhanced visual guidance, enables less-experienced operators to perform AI more efficiently and accurately. From an economic perspective, VE-TCAI offers a cost-effective alternative to LAI, as it requires only a portable endoscopic system rather than surgical instruments or anesthesia. The endoscope used in this study has been in continuous operation for more than two years without technical failure, demonstrating its durability and suitability for routine field application. Although initial investment and basic operator training may represent minor barriers, VE-TCAI can be readily integrated into community-based or smallholder breeding programs as a minimally invasive, rapid, and efficient reproductive management tool. With basic restraint and limited training, AI service providers can effectively perform insemi-nations under typical field conditions. Nevertheless, strict biosecurity measures, including thorough cleaning and disinfection of endoscopic equipment between animals, are essential to prevent cross-contamination and ensure hygienic application [21].

Despite the significant reduction in insemination time, pregnancy rates did not differ statistically between the two methods (p = 0.737), although they were numerically higher in the VE-TCAI group (45.5%) than in the C-TCAI group (33.3%). This finding suggests that factors beyond insemination duration also influence pregnancy outcomes [26]. Environmental stressors, including high ambient temperature, humidity, and nutritional fluctuations during the breeding season, may impair oocyte quality, hormonal balance, and uterine receptivity, thereby reducing conception efficiency under tropical conditions. The abortion and maternal death observed in Farm 2 may reflect the adverse effects of heat and metabolic stress on placental function and fetal survival in small ruminants [27]. Consequently, the lack of statistical significance in pregnancy rates is more likely attributable to the limited sample size rather than the absence of a biological effect, given the consistent trend toward higher fertility in the VE-TCAI group.

Unlike earlier studies conducted in experimental stations or focused on pure breeds, this study demonstrates the feasibility of VE-TCAI under field conditions in native–Boer crossbred goats, a production-relevant genotype in tropical systems where LAI is often impractical. To the best of our knowledge, this is the first study to quantify insemination duration as an operational performance indicator in goats and to propose a ≤1-min benchmark for evaluating procedural efficiency under field conditions. The approximately 40% reduction in insemination time achieved with VE-TCAI highlights its potential as a measurable efficiency parameter in reproductive biotech-nology. Although correlation or regression analyses were not feasible due to the categorical data structure and limited sample size, the observed trend linking shorter insemination times with higher conception success suggests a mechanistic relationship between procedural efficiency and fertility outcomes. This observation is consistent with previous evidence indicating that precise intrauterine or deeper transcervical semen deposition improves fertility by enhancing sperm transport and reducing semen loss [16, 25, 28].

Estrus synchronization protocols and the timing of insemination are critical determinants of pregnancy success. In the present study, synchronization using a CIDR device combined with eCG and Prostaglandin F₂ alpha proved effective for fixed-time AI. A 48-h interval after CIDR removal was selected based on pre-study observations and was considered to fall within the ovulatory window. Although earlier studies suggested an optimal insemination window of 56–60 h [8, 29], breed-specific physiology, environmental conditions, and hormonal regimens may have influenced ovulation timing, allowing satisfactory conception at the shorter interval. Insemination conducted 12–24 h after the onset of standing estrus is known to yield higher pregnancy rates, and multiple inseminations within the same estrus may further improve outcomes by ensuring the presence of viable spermatozoa at ovulation. Hormonal protocols incorporating eCG can enhance estrus expression and ovulation synchronization, thereby supporting AI success [30, 31].

Although VE-TCAI requires a higher initial investment than conventional AI equipment, its potential to improve efficiency and reproductive outcomes may render it economically viable for medium- to large-scale goat operations [1]. Nonetheless, strict sanitation and sterilization protocols remain essential, as inadequate cleaning of endoscopic equipment increases the risk of pathogen transmission [21].

The lack of blinding during pregnancy diagnosis may have introduced observer bias, despite the use of objective ultrasonographic criteria. In addition, no adjustment for multiple comparisons was applied, which may slightly increase the risk of type I error. Future studies should incorporate larger sample sizes, blinded assessments, and multi-farm designs to validate these findings. The integration of endoscopic systems with real-time ovulation monitoring may further optimize conception outcomes in field settings.

Beyond its immediate application in AI, VE-TCAI may serve as a foundational platform for advancing reproductive biotechnology in small ruminants. Its minimally invasive nature and real-time visual access make it suitable for future integration with non-surgical embryo transfer, genetic improvement programs, and synchronization-based breeding strategies. Furthermore, coupling VE-TCAI with real-time ovulation monitoring or AI-assisted image analysis for cervical navigation could enable precision timing and scalable reproductive management under field conditions.