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This work was supported by the Utah Agricultural Experiment Station (UAES). The authors gratefully acknowledge UAES for financial support and thank the personnel and veterinary clinic staff at Utah State University South Farm for their technical assistance throughout this study.

<ol>
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The authors declare that they have no competing financial interests.

The present study indicates that the described synchronization and superstimulation protocol, combined with LOPU, supports ovarian response and efficient in vivo oocyte recovery in alpacas. Animals treated with 200 mg FSH developed a substantial follicular population, with approximately three-quarters of follicles successfully aspirated and a recovery rate exceeding 60%. All visible follicles greater than 2 mm in diameter that could be adequately stabilized were aspirated; however, a proportion of follicles remained inaccessible, likely due to insufficient firmness of ovarian fixation during the procedure, which may have limited complete needle access to all visible follicles. These outcomes nonetheless indicate that the hormonal regimen effectively induces follicular development while maintaining procedural efficiency during follicular aspiration.
GnRH administration at the time of dominant follicle detection has become the preferred synchronization approach in non-mated alpacas, given that during their prolonged follicular phase, dominant follicles repeatedly reach diameters sufficient to respond to exogenous GnRH, enabling reliable follicular wave turnover34,35,36,37. While progesterone-based devices and prostaglandin protocols have been explored in Alpacas, these approaches have yielded inconsistent results due to device expulsion, vaginal complications, and unreliable follicular suppression35, supporting the selection of the GnRH-based protocol used in the present study for its practicality and compatibility with LOPU for consistent oocyte recovery in alpacas.
The predominance of medium- to large-sized follicles (5&#8211;6 mm) at the time of collection is consistent with an active follicular response to the superstimulation protocol. In alpacas, follicle size has been associated with in vitro cytoplasmic maturation and developmental potential38, suggesting that the follicular profile observed may favor the recovery of morphologically suitable cumulus&#8211;oocyte complexes (COCs), as illustrated in Figure 5. However, as the present study was not designed to directly compare FSH dosing regimens and the sample size was limited, future studies with larger animal numbers and comparative treatment designs would be needed to confirm whether the decreasing-dose FSH protocol specifically promotes superior follicular synchrony or oocyte quality in alpacas.
The morphological diversity observed among recovered oocytes reflects the inherent biological variability of superstimulation responses. High-grade oocytes exhibited compact, multilayered cumulus investment and homogeneous cytoplasm, features commonly associated with improved in vitro maturation and embryo development potential. In contrast, lower-grade oocytes displayed partial cumulus loss or cytoplasmic irregularities, which may compromise further developmental competence39. Nonetheless, the overall proportion of recovered oocytes and the consistency of follicular response indicate that the protocol provides a reliable source of in vivo&#8211;derived COCs suitable for downstream applications.
Almost 60% of the recovered oocytes were classified as Grade I&#8211;II, representing a substantial proportion of morphologically suitable oocytes for in vitro procedures, particularly in settings where slaughterhouse-derived ovaries are unavailable or limited. In contrast, an important proportion of the recovered oocytes were classified as Grade III&#8211;IV, characterized by partial or complete absence of cumulus cell investment. This may be partially attributed to the aspiration conditions of the vacuum pump, including pressure settings, needle diameter, tubing length, and turbulence generated during follicular aspiration, as previously reported for oocyte collection procedures in other species29. Optimization of these aspiration parameters may therefore represent an important consideration for improving oocyte morphological quality in future LOPU procedures in alpacas.
The aspiration flow rate represents a critical technical parameter during follicular oocyte recovery, as excessive pump speeds have been associated with mechanical disruption of the cumulus&#8211;oocyte complex (COCs) and a consequent reduction in oocyte morphological quality. With a constant-flow pump system to retrieve oocytes from ovine ovaries obtained from slaughterhouse, flow rates exceeding 20 mL/min were reported to be detrimental to oocyte morphological integrity, with an optimal range of 10&#8211;20 mL/min recommended to minimize aspiration-induced damage41 . In contrast, earlier LOPU studies conducted across various species have described aspiration rates of 50&#8211;70 drops per minute as operationally effective10,12,13,14,15,16 . More recently, an aspiration flow rate of 22 mL/min during OPU with transvaginal ultrasound-guided follicle aspiration was reported to result in marked morphological compromise of oocytes recovered in alpacas, further supporting the use of lower flow rates to preserve COCs integrity7. There is limited peer-reviewed literature on specific aspiration flow rate parameters for LOPU in alpacas.
In the present study, although a systematic comparison of aspiration flow rates was not conducted, a flow rate of 13&#8211;15 mL/min at a negative pressure of 30 mmHg was selected as the operative setting based on preliminary testing of multiple flow rate conditions. This range falls within the optimal interval described for sheep41 and is below the threshold associated with oocyte morphological deterioration in alpacas7. No apparent adverse effects on COCs morphology were detected under these conditions, suggesting that the operative parameters employed were appropriate. Future studies by systematically evaluating a broader range of aspiration flow rates under LOPU conditions in this species are warranted to establish evidence-based standardized protocols. LOPU has been demonstrated in small ruminants, including goats and sheep, to be a safe and repeatable procedure that can be performed multiple times in the same animal without inducing significant pain, postoperative complications, or detrimental effects on reproductive performance23,40. However, in alpacas, the impact of repeated oocyte collection on animal welfare and subsequent reproductive efficiency has not been systematically evaluated. Although the present study was not specifically designed to assess these effects, it is worth noting that LOPU was performed 10 times on each animal over a 10-month period, and no adhesions or morphological alterations of the reproductive organs or abdominal wall were observed throughout the study. These observations are descriptive and were not derived from a study specifically designed to evaluate the long-term effects of repeated LOPU on reproductive performance or animal welfare. Systematic studies are nonetheless necessary to formally evaluate these variables in alpacas and determine whether repeated LOPU adversely affects short- and long-term reproductive performance under the conditions described.
Compared to ultrasound-guided ovum pick-up approaches, LOPU offers direct ovarian visualization and may enhance follicular access in alpacas, whose anatomical characteristics can limit the effectiveness of transvaginal techniques9. OPU in alpacas has been reported to achieve collection rates of approximately 75%, recovering a mean of 3.3 oocytes per ovary, with oocytes classified as Grade I and II representing only 30% of the total recovered7. In contrast, the LOPU protocol described in the present study yielded a collection rate of 61%, while the proportion of morphologically suitable oocytes classified as Grade I and II reached approximately 60% of total recovered oocytes, a substantially higher proportion than that reported with OPU.
Although the collection rate observed with LOPU was numerically lower than that reported for transvaginal OPU, the marked improvement in oocyte morphological quality suggests that LOPU may represent a more advantageous approach for downstream in vitro embryo production applications in alpacas. Both techniques share susceptibility to aspiration-related morphological compromise, likely attributable to vacuum pressure and flow rate conditions during follicular aspiration7,8. Notably, oocyte recovery rates obtained with LOPU in alpacas are comparable to those reported in small ruminants9,10,27, collectively demonstrating that laparoscopic retrieval is both feasible and reproducible in this species when appropriate hormonal preparation and technical expertise are applied. Several technical factors may influence oocyte harvest and quality outcomes, including follicular wave synchronization accuracy, timing of aspiration relative to the final FSH dose, surgeon experience, and aspiration system parameters such as vacuum pressure and flow rate, length of the aspiration line, tubing diameter, needle diameter, and the turbulence generated during the alternating suction and non-suction cycles of the pump41. Standardization of these parameters is essential to optimize oocyte harvest and morphological quality. Additionally, while morphological assessment provides a practical measure of oocyte evaluation, further studies incorporating in vitro maturation, fertilization, and embryo development endpoints would strengthen the functional validation of the protocol and confirm the developmental potential of oocytes recovered through LOPU in alpacas.
In conclusion, the combined application of GnRH-induced synchronization, decreasing-dose FSH superstimulation, and LOPU appeared to represent a feasible strategy for in vivo oocyte recovery in alpacas under the conditions described. The follicular response, aspiration efficiency, and oocyte recovery rates observed suggest that this protocol may be suitable for application in reproductive biotechnology programs in this species. No apparent adverse effects on ovarian integrity or donor health were observed across repeated procedures; however, systematic evaluation of the long-term impact of repeated LOPU sessions on reproductive performance in alpacas is still needed. These findings may contribute to the growing body of evidence supporting the application of assisted reproductive technologies in South American camelids and could serve as a preliminary foundation for future investigations into in vitro embryo production, embryo transfer, and related germline-based procedures in alpacas, provided that studies incorporating larger animal numbers are conducted to generate more robust and generalizable data.
Limitations 
The present study has limitations that should be acknowledged. First, the relatively small number of donor animals limits the statistical power of the findings and may not fully capture the biological variability inherent to the alpaca population. Second, oocyte quality was assessed solely by morphological classification, without functional validation of developmental competence via IVM, IVF, and IVC. Morphological grading, while practical and widely used, does not necessarily reflect the true developmental potential of recovered oocytes. Future studies incorporating these in vitro endpoints are therefore essential to further evaluate the protocol and to confirm the functional suitability of LOPU-derived oocytes for embryo production and advanced reproductive biotechnology applications in alpacas.

South American camelids, particularly alpacas (Vicugna pacos), have gained increasing relevance in reproductive biotechnology due to their distinctive reproductive physiology and expanding role in biomedical and agricultural research. Alpacas exhibit induced ovulation, prolonged follicular waves, and unique ovarian dynamics that differ markedly from those of domestic ruminants1. These characteristics, together with the growing economic and scientific importance of the species, have driven efforts to develop assisted reproductive technologies (ARTs) tailored to camelid biology. Nevertheless, progress in alpaca ARTs has historically lagged behind that achieved in cattle, sheep, and goats, largely owing to anatomical constraints, limited access to high-quality gametes, and technical challenges associated with in vivo oocyte recovery2&#722;3.
Early attempts to recover oocytes from live alpacas relied primarily on surgical laparotomy, a method adapted from foundational studies in South American camelid embryo research3&#722;4. Although laparotomy enabled direct visualization and aspiration of ovarian follicles, it was highly invasive, required extended postoperative recovery, and restricted the frequency of donor use. Consequently, this approach proved unsuitable for repeated collections or large-scale embryo production programs.
Subsequent advances led to the development of ultrasound-guided ovum pick-up (OPU) techniques, enabling minimally invasive oocyte collection and reducing surgical intervention5,6,7,8. While this approach improved animal welfare and procedural efficiency, the relatively small body size and anatomical constraints of alpacas may limit probe positioning and follicle accessibility, potentially affecting consistency and recovery efficiency1. Moreover, successful implementation requires specialized personnel training to ensure appropriate equipment handling, accurate follicular visualization, and reproducible outcomes7&#722;8.
Laparoscopic ovum pick-up (LOPU) has emerged as a significant advancement in assisted reproduction. Initially established in small ruminants9.10.11, it has since been adapted across a broad range of species, including prepubertal cattle12&#8211;14, deer15&#722;16, pigs17&#722;18, felids (e.g., pumas, jaguars, and tigers)19&#722;20, and non-human primates21&#722;22, highlighting its versatility across domestic and wildlife species. The technique provides direct transabdominal access to the ovaries through minimally invasive entry, enabling precise follicular aspiration under real-time visual guidance9&#722;23. Although LOPU has been extensively refined in prepubertal cattle12&#8211;14, sheep10&#722;11&#722;23, and goats11&#722;24&#8211;27, its application in alpacas (Vicugna pacos) remains comparatively limited28. Nevertheless, available evidence suggests that LOPU can achieve outcomes comparable to those reported in small ruminants while avoiding the need for rectal manipulation, supporting its potential as a technically robust approach for oocyte collection in this species29&#722;30.
Despite these advances, standardized and reproducible LOPU protocols for alpacas remain scarce. Most camelid ARTs studies have focused on in vitro maturation, fertilization, and embryo culture using oocytes derived from slaughterhouse ovaries or ultrasound-guided aspiration8&#722;31&#722;32. Although these approaches have provided valuable insights into oocyte competence and embryo development, they do not ensure a consistent supply of high-quality, in vivo&#8211;derived oocytes from genetically valuable donors. Furthermore, recent studies have highlighted the influence of the collection method on oocyte quality, underscoring the need for optimized in vivo retrieval techniques7.
The present study provides a detailed, step-by-step description of a laparoscopic ovum pick-up procedure specifically adapted for alpacas. This work is intended as a procedural and technical report rather than a comprehensive biological validation study. The protocol integrates current knowledge of camelid reproductive physiology, established principles from LOPU in small ruminants, and practical considerations related to anesthesia, ovarian superstimulation, laparoscopic access, and follicular aspiration. By presenting a reproducible and minimally invasive approach, this study aims to support the broader adoption of LOPU in alpaca reproductive programs and to facilitate future applications in in vitro embryo production, embryo transfer, and genetic engineering.

<table><tbody><tr><td>Ampicillin</td><td>Valleyvet</td><td>1787RX</td><td>Ampicillin Powder for Injectable Suspension 25 mg</td></tr><tr><td>Aspiration Needle&amp;nbsp;</td><td>WTA Technologies</td><td>26041</td><td>20G</td></tr><tr><td>Aspiration Pump for bovine OPU</td><td>Minitube</td><td>23362/0001</td><td>Warming block, foot pedal, 115 V</td></tr><tr><td>Babcock Forceps</td><td>Karl Storz</td><td>26166</td><td>5 mm, 30 cm&amp;nbsp;</td></tr><tr><td>Blunt Cannula</td><td>Medline</td><td>MDS0775641</td><td>23 G</td></tr><tr><td>Camera Head</td><td>ESC Medicams</td><td>ESC2000212</td><td>HD, portable endoscopy camera</td></tr><tr><td>Carbon Dioxide</td><td>Airgas</td><td>N/A</td><td>20-pound cylinder, USP Medical, CGA 320 washer</td></tr><tr><td>Chlorhexidine 2% solution</td><td>Durvet</td><td>7-45801-10259-0</td><td>N/A</td></tr><tr><td>Cradle for laparoscope</td><td>Custom-built at Utah State University</td><td>N/A</td><td>Inclination range of 30&amp;ndash;45&amp;deg;, with extended support attachment for the alpaca neck</td></tr><tr><td>Disposable Scalpel Blades</td><td>Medline</td><td>CISION22SS</td><td>Stainless-Steel Blade, No. 22. Sterile</td></tr><tr><td>Disposable Skin Stapler</td><td>Medline</td><td>QTX253001H</td><td>35 mm</td></tr><tr><td>Dulbecco&#039;s Phospate Buffered Saline</td><td>Gibco</td><td>14190144</td><td>No Calcium, No Magnesium</td></tr><tr><td>Endotracheal tube</td><td>Penn Veterinary Supply, Inc</td><td>JORJ615-H</td><td>43Fr 10.0 mm silicone cuffed</td></tr><tr><td>Estroplan</td><td>AmerisourceBergen</td><td>52077</td><td>Cloprostenol 250 mg/mL</td></tr><tr><td>Fertagyl</td><td>AmerisourceBergen</td><td>60654</td><td>Gonadorelin 43mg/mL&amp;nbsp;</td></tr><tr><td>Fiber-Optic Light Cable</td><td>Olympus</td><td>WA03210A</td><td>5 mm&amp;nbsp;</td></tr><tr><td>Folltropin Dual Pack</td><td>Professional Embryo Transfer Supply, Inc</td><td>04-020-129-3</td><td>Porcine Pituitary-Derived Follicle Stimulating Hormone 700 IU FSH</td></tr><tr><td>Halogen Ligh Source</td><td>Olympus</td><td>CLC-SC</td><td>Halogen Lamp, 120 V, 1.7A</td></tr><tr><td>Heparin Sodium Salt</td><td>Sigma-Aldrich</td><td>H4784</td><td>&amp;ge;180 USP units/mg</td></tr><tr><td>Insuffation Tubing</td><td>Medline</td><td>DYNJ05933</td><td>Luer, Sterile</td></tr><tr><td>Isoflurane vaporaizer</td><td>VetEquip</td><td>911103</td><td>Equipped with scavenging collection bag</td></tr><tr><td>Ketamine</td><td>Dechra</td><td>1000001250</td><td>Ketamine Hydrochloride 100 mg/mL</td></tr><tr><td>Lubricant</td><td>Durvet</td><td>7-45801-11406-7</td><td>All purpose&amp;nbsp;</td></tr><tr><td>Medium 199</td><td>Gibco</td><td>11150059</td><td>Earle&#039;s Salt, No suplemented</td></tr><tr><td>Meloxicam</td><td>Valleyvet</td><td>1545RX</td><td>Meloxicam tablets,&amp;nbsp; USP 15 mg</td></tr><tr><td>Monitor series S32GF</td><td>Samsung</td><td>&amp;lrm;LS24F320GANXZA</td><td>FHD, 24 inches</td></tr><tr><td>Rigid Laparoscope</td><td>Olympus</td><td>A50372A</td><td>5 mm, 0&amp;deg;, 30 cm</td></tr><tr><td>Rompun</td><td>Dechra</td><td>1000001151</td><td>Xylazine 100 mg/mL</td></tr><tr><td>SAS software</td><td>SAS Institute Inc.</td><td>N/A</td><td>Used for descriptive statistical analysis</td></tr><tr><td>Scalpel Handle&amp;nbsp;</td><td>Medline</td><td>MDS10801</td><td>#3</td></tr><tr><td>Stereo Microscope</td><td>Olympus</td><td>SZ61</td><td>Transmitted light mirrored base</td></tr><tr><td>Sterile Disposable Drapes</td><td>Medline</td><td>LPC980927</td><td>N/A</td></tr><tr><td>Torphadine</td><td>Dechra</td><td>1000001776</td><td>Butorphanol Tartrate 10 mg/mL</td></tr><tr><td>Trocar-Cannula</td><td>Endoscopy Superstore</td><td>101021-A</td><td>5 mm, Spiral, with automatic valve</td></tr><tr><td>Ultrasound Diagnostic System&amp;nbsp;</td><td>Chison</td><td>Eco1</td><td>Lineal Probe 7.5 MHz</td></tr></tbody></table>

laparoscopic ovum pick-up for in vivo oocyte retrieval in alpacas

Laparoscopic Ovum Pick-up For In Vivo Oocyte Retrieval In Alpacas

Alpacas are increasingly incorporated into reproductive and biomedical research due to distinctive physiological and immunological characteristics that extend their relevance beyond agricultural applications. As camelids, alpacas exhibit induced ovulation, prolonged follicular dynamics, asymmetric uterine physiology, and a unique immune system characterized by heavy-chain&#8211;only antibodies. These features support their use in comparative and translational research, particularly in reproductive biology and genetic engineering. Laparoscopic ovum pick-up (LOPU) is a minimally invasive approach for oocyte recovery in species with complex reproductive anatomy, allowing direct ovarian visualization, precise follicular aspiration, and reduced tissue trauma compared with transvaginal techniques. The objective of this study was to develop and standardize a reproducible in vivo oocyte retrieval protocol in alpacas using LOPU. Eight adult female alpacas (3&#8211;8 years of age) were enrolled in this study and subjected to repeated LOPU sessions conducted once per month throughout the study period. Alpacas were evaluated by transrectal ultrasonography, and animals presenting ovarian follicles &#8805;7 mm received 50 &#181;g gonadotropin-releasing hormone intramuscularly. Ovarian superstimulation was induced using a total dose of 200 mg follicle-stimulating hormone administered in a decreasing twice-daily regimen over four days. LOPU was performed 12&#8211;14 h after the final hormone administration under general anesthesia induced with ketamine, xylazine, and butorphanol and maintained with isoflurane. A three-port laparoscopic approach with carbon dioxide insufflation was used. Follicles were aspirated using a 20-gauge needle connected to a regulated vacuum system (25&#8211;30 mmHg; 13&#8211;15 mL/min). Oocytes were recovered in TCM-199 medium and evaluated under a stereomicroscope. Alpacas exhibited a mean of 14.88 &#177; 3.85 follicles, with 10.94 &#177; 3.62 follicles aspirated per session and 6.68 &#177; 2.21 oocytes recovered, corresponding to a recovery rate of 61.30 &#177; 8.21%. These findings suggest that LOPU may represent a viable approach for in vivo oocyte retrieval in alpacas, with potential applications in reproductive biotechnology workflows.

Ovarian response and oocyte recovery following FSH superstimulation in alpacas are summarized in Table 1. Animals synchronized and superstimulated with 200 mg FSH developed an average of 14.88 &#177; 3.85 ovarian follicles per session, of which 10.94 &#177; 3.62 were successfully aspirated, resulting in a follicle aspiration rate of 74.18 &#177; 8.91%. A mean of 6.68 &#177; 2.21 oocytes were recovered per procedure, corresponding to an oocyte recovery rate of 61.30 &#177; 8.21%. Of the total oocytes collected, 59.05% were classified as Grade I&#8211;II, representing a substantial proportion of oocytes meeting morphological criteria for further use in in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro culture (IVC) protocols.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Total Ovarian Follicles (n)</td>
			<td rowspan="2">Ovarian Follicles Aspirated (n)</td>
			<td rowspan="2">Follicle Aspiration Rate (%)</td>
			<td rowspan="2">Oocytes Collected (n)</td>
			<td rowspan="2">Oocyte Recovery Rate (%)</td>
			<td colspan="4">Oocyte Morphological Classification (%)</td>
		</tr>
		<tr>
			<td>G1</td>
			<td>G2</td>
			<td>G3</td>
			<td>G4</td>
		</tr>
		<tr>
			<td>14.88 &#177; 3.85</td>
			<td>10.94 &#177; 3.62</td>
			<td>74.18 &#177; 8.91</td>
			<td>6.68 &#177; 2.21</td>
			<td>61.30 &#177; 8.21</td>
			<td>48.57</td>
			<td>10.48</td>
			<td>26.67</td>
			<td>14.29</td>
		</tr>
		<tr>
			<td colspan="9">Values are presented as mean &#177; standard deviation SD.</td>
		</tr>
	</tbody>
</table>
Table 1: Ovarian response and oocyte recovery following FSH superstimulation and laparoscopic ovum pick-up (LOPU) in alpacas. Total ovarian follicles, ovarian follicles aspirated, and oocytes collected are expressed as averages across all collection sessions. Follicle aspiration rate, oocyte recovery rate, and oocyte morphological classification are expressed as percentages. Oocytes were morphologically classified as follows: Grade I (G1) &#8212; at least two complete compact cumulus cell layers and homogeneous cytoplasm; Grade II (G2) &#8212; at least one intact cumulus layer with mostly homogeneous cytoplasm; Grade III (G3) &#8212; partial or incomplete cumulus layers with cytoplasmic heterogeneity; and Grade IV (G4) &#8212; absent cumulus investment with fragmented or degenerated cytoplasm. Values are presented as mean &#177; standard deviation (SD), where applicable.
Representative laparoscopic images illustrating ovarian morphology and structures observed during LOPU are shown in Figure 3. Ovaries exhibiting a strong follicular response contained multiple well-developed follicles larger than 5&#8211;6 mm in diameter (Figure 3A&#8211;D). Following follicular aspiration, ovaries displayed reduced follicular turgidity and visible puncture sites corresponding to aspirated follicles (Figure 3E&#8211;H). In contrast, some animals exhibited limited follicular development, characterized by a small number of follicles and reduced ovarian activity (Figure 3I&#8211;L). Additional ovarian structures were occasionally observed during the procedure, including follicular cysts (FC) (Figure 3M, N). Finally, corpora lutea (CL) were observed as an indication of recent ovulation (Figure 3O, P).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/71397/71397fig3.jpg" /> 
Figure 3: Representative laparoscopic images of ovarian morphology and associated structures observed during LOPU in alpacas. Panels A&#8211;D illustrate a strong follicular response with multiple well-developed follicles (5&#8211;6 mm in diameter). Panels E&#8211;H show ovaries following follicular aspiration, with visible puncture sites and reduced follicular turgidity. Panels I&#8211;L depict limited follicular development representative of a poor ovarian response. Panels M&#8211;N show a follicular cyst (FC). Panel O shows a corpus luteum (CL). Panel P shows the right uterine horn and associated ovary. Scale bars = 5 mm. <a href="https://www.jove.com/files/ftp_upload/71397/71397fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Descriptive analysis of follicular size distribution revealed a predominance of medium- to large-sized follicles at the time of collection (Figure 4). Follicles measuring 5&#8211;6 mm in diameter constituted a greater proportion of the ovarian follicular population compared with follicles measuring 3&#8211;4 mm. In contrast, the smaller follicle category (3&#8211;4 mm) exhibited greater variability and represented a lower percentage of the total follicular pool. These observations indicate that the superstimulation protocol promoted follicular development within the size range considered suitable for oocyte aspiration.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/71397/71397fig4.jpg" /> 
Figure 4: Ovarian follicle size distribution at Laparoscopic Ovum Pick-up (LOPU) in FSH-superstimulated alpacas. Box plots illustrate the percentage distribution of follicles within two diameter classes (3&#8211;4 mm and 5&#8211;6 mm) recovered during laparoscopic oocyte pick-up (LOPU) following GnRH synchronization and FSH superstimulation. The box represents the interquartile range (IQR; 25th&#8211;75th percentiles), the horizontal line within the box indicates the median, whiskers extend to minimum and maximum values, and the circle (&#9675;) denotes the group mean. <a href="https://www.jove.com/files/ftp_upload/71397/71397fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Representative images of cumulus&#8211;oocyte complexes (COCs) and oocytes collected by laparoscopic ovum pick-up (LOPU) are shown in Figure 5. The micrographs illustrate variations in morphological quality, including well-preserved cumulus investment and homogeneous cytoplasm in high-grade oocytes, and reduced cumulus coverage and cytoplasmic heterogeneity in lower-grade oocytes.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/71397/71397fig5.jpg" /> 
Figure 5: Representative micrographs of immature cumulus&#8211;oocyte complexes (COCs) recovered from alpacas (Vicugna pacos) by laparoscopic ovum pick-up (LOPU) and classified according to morphological quality. Upper panels illustrate Grade I and II oocytes, characterized by compact cumulus cell investment and homogeneous cytoplasm. Lower panels show Grade III and IV oocytes, exhibiting partial or complete absence of cumulus cells and cytoplasmic irregularities. Scale bars = 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/71397/71397fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Collectively, the observed follicular response, aspiration efficiency, and oocyte recovery rates observed in the present study suggest that the hormonal synchronization and superstimulation regimen evaluated may support in vivo oocyte collection in alpacas. The proportion of aspiratable follicles and the recovery of morphologically suitable oocytes indicate that this protocol may be applicable to subsequent in vitro embryo production, embryo transfer, and related reproductive biotechnology procedures in this species, although studies with a larger number of animals are needed to strengthen and validate these findings.

This protocol describes a reproducible method for in vivo oocyte recovery in alpacas using hormonal synchronization, FSH superstimulation, and laparoscopic ovum pick-up (LOPU). The procedure yields consistent follicular responses and efficient oocyte recovery, supporting applications for in vitro embryo production and advanced reproductive biotechnologies.

Alpacas are increasingly incorporated into reproductive and biomedical research due to distinctive physiological and immunological characteristics that ...

My research focuses on improving assisted reproductive technologies in alpacas, specifically optimizing oocyte collection through laparoscopic one pickup. Transvaginal approaches are limited in alpaca due to anatomical constraints. This laparoscopic protocol provides a more effective and reliable alternative.To begin, position the anesthetized and intubated Huacaya female alpaca on a gurney bed. Review the animal's treatment record to confirm that the alpaca has undergone follicular wave synchronization with GnRH and ovarian super stimulation with FSH before oocyte collection. Shave the abdominal region.Thoroughly cleanse the area with surgical soap. Disinfect with ethanol. And apply an iodine-based antiseptic solution.Transfer the animal to the designated surgical area and secure it in place while maintaining proper alignment and aseptic conditions. Cover the abdominal region with a sterile surgical drape and create a fenestration to expose only the operative field. Using a scalpel, make a midline skin incision approximately 10 to 15 centimeters cranial to the udder.Introduce a blunt insufflation needle into the abdominal cavity. Insufflate the abdominal cavity with carbon dioxide until adequate abdominal distension is achieved. Insert a five-millimeter valved trocar through the initial incision to access the abdominal cavity and introduce the laparoscopic video camera.Place two additional five-millimeter valved trocars, one on each side of the first port, 10 centimeters lateral to the midline and five centimeters cranial to the udder. Place the female alpaca in the Trendelenburg position. With atraumatic forceps, gently manipulate and mobilize the ovary to optimize follicle exposure and release it from the infundibulum.Prepare a single lumen aspiration system comprising a 20-gauge short bevel needle connected to a 50-centimeter cannula, a silicone stopper, and a 50 milliliter collection tube prefilled with collection medium. After connecting the aspiration pump, set it to a vacuum pressure of 25 to 30 millimeters of mercury and a flow rate of 13 to 15 milliliters per minute. Introduce atraumatic Babcock forceps through one port and gently stabilize the ovary at the mesovarium.Insert the aspiration needle through the second port. Puncture each follicle and gently rotate the needle to aspirate the follicular fluid while minimizing disruption of the cumulus oocyte complexes. Using a 10-milliliter syringe introduced through the cannula, gently maneuver the ovary toward the syringe while moving the cannula over the ovarian surface.Flush with warm sterile saline solution containing 0.1 milligrams per milliliter heparin and gently agitate to remove residual blood and minimize adhesion formation. Repeat the procedure on the contralateral side and then decompress the abdomen. Close the skin incision with surgical staples.Dry the surgical wound with sterile gauze. Apply a topical repellent and healing ointment. If a corpus luteum is identified during the procedure, administer two milliliters of 250 micrograms per milliliter cloprostenol intramuscularly.At the end of the procedure, reduce the vaporizer setting to 0%and maintain oxygen flow to facilitate anesthetic washout until the animal exhibits signs of recovery. Monitor the animal during recovery by assessing pulse oximetry, body temperature, respiratory effort and overall behavior. Discontinue the oxygen supply following completion of anesthetic recovery.Using a 20-milliliter syringe, deflate the endotracheal tube cuff and carefully remove the endotracheal tube once protective reflexes return. Using a trimmed one-milliliter pipette tip, transfer the follicular fluid and collection medium from the 50 milliliter conical tube into a sterile Petri dish. Examine the contents under a stereo microscope to identify cumulus oocyte complexes.Using a sterile pipette, recover oocytes from the medium. Transfer the oocytes into maturation medium and grade them according to morphological characteristics. Animals synchronized and super stimulated with FSH developed a mean of 14.88 ovarian follicles per session.Of these, 10.94 were aspirated, corresponding to a follicle aspiration rate of about 74.18%A mean of 6.68 oocytes was recovered per procedure, corresponding to an oocyte recovery rate of about 61.30%Of the total oocytes collected, 59.05%were classified as grade 1 to 2. Follicles measuring five to six millimeters in diameter constituted a greater proportion of the ovarian follicular population compared with follicles measuring three to four millimeters. The micrographs illustrated well-preserved cumulus investment and homogeneous cytoplasm in the grade 1 and 2 oocytes.Reduced cumulus coverage and cytoplasmic heterogeneity were observed in the grade 3 and 4 oocytes. This protocol enables oocyte yield assessment and provides materials for downstream procedures, including IVF and genetic engineering in alpacas. Correctly synchronizing the follicular wave and carefully managing the factor that affect the aspiration efficiency is the most critical steps.Future studies can optimize in vitro maturation, fertilization and gene editing protocol to advance in reproductive technologies in alpacas.

laparoscopic-ovum-pick-up-for-in-vivo-oocyte-retrieval-in-alpacas

Research

JoVE Journal

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Utah State University.  This protocol describes a reproducible method for in vivo oocyte recovery in alpacas using hormonal synchronization, FSH superstimulation, and laparoscopic ovum pick-up (LOPU). The procedure yields consistent follicular responses and efficient oocyte recovery, supporting applications for in vitro embryo production and advanced reproductive biotechnologies.

Laparoscopic Ovum Pick-up For In Vivo Oocyte Retrieval In Alpacas

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