Kualitas dan Freezability Spermatozoa Domba Lokal Indonesia dalam Berbagai Pengencer

Abstract

Penurunan populasi domba lokal Indonesia menuntut penerapan teknologi reproduksi untuk mempertahankan produktivitas dan meningkatkan keberhasilan inseminasi buatan (IB). Keberhasilan IB sangat bergantung pada kualitas semen, khususnya kemampuan spermatozoa bertahan selama proses pembekuan (freezability). Kualitas semen beku salah satunya dipengaruhi oleh bahan pengencer yang berfungsi melindungi membran spermatozoa terhadap stres kriogenik. Pengencer semen terdiri atas pengencer buatan sendiri (home made) dan pengencer komersial. Penelitian ini dilakukan untuk menganalisis pengaruh pengencer homemade dan komersial terhadap kualitas, kinematik, longivitas, serta recovery rate (RR) spermatozoa semen beku domba lokal yang sebagian besar menggunakan pendekatan objektif dengan Computer Assisted Sperm Analysis (CASA). Penelitian menggunakan tiga individu domba lokal dengan enam kali pengulangan. Evaluasi dilakukan terhadap semen segar dan semen beku menggunakan empat jenis pengencer, yaitu Tris Kuning Telur (TKT, homemade), AndroMed®, BoviFree®, dan Steridyl®. Parameter yang diamati meliputi karakteristik makroskopis, motilitas, viabilitas, integritas akrosom, abnormalitas, konsentrasi spermatozoa, serta variabel kinematik spermatozoa yakni curvilinear velocity (VCL), straight-line velocity (VSL), average pathway velocity (VAP), amplitude of lateral head displacement (ALH), beat cross frequency (BCF), linearity (LIN), and straightness (STR). Hasil penelitian menunjukkan karakteristik semen segar makroskopis normal dengan volume rata-rata 1,32 ± 0,13 mL, warna krem, konsistensi kental, dan pH 6,64 ± 0,05. Motilitas total dan progresif masing-masing sebesar 68,11 ± 3,83% dan 62,09 ± 4,07%, dengan viabilitas 53,61 ± 3,57%, integritas akrosom 95,22 ± 1,14%, dan abnormalitas 23,04 ± 2,64%. Konsentrasi spermatozoa rata-rata 2.754,65 ± 178,94 ×106/mL (metode CASA). Evaluasi kinematik menunjukkan nilai VCL 198,82 ± 8,47 µm/s, VSL 88,80 ± 5,84 µm/s, VAP 103,25 ± 6,12 µm/s, ALH 3,88 ± 0,11 µm, BCF 12,73 ± 0,45 Hz, LIN 0,46 ± 0,01, dan STR 0,83 ± 0,01. Nilai-nilai tersebut menggambarkan aktivitas spermatozoa yang tinggi, gerakan yang stabil, serta potensi fertilitas yang baik. Spermatozoa setelah pembekuan mengalami penurunan kualitas pada semua pengencer. Spermatozoa dalam pengencer Steridyl® menunjukkan motilitas total tertinggi (44,98 ± 4,72%), motilitas progresif (41,07 ± 4,56%), dan viabilitas (42,56 ± 4,35%) yang melebihi persyaratan mutu minimal Standar Nasional Indonesia (SNI) 4869-3:2023 untuk semen beku domba dan kambing (=40%). Pengencer AndroMed® dan BoviFree® menunjukkan performa sedang, sedangkan TKT menghasilkan nilai terendah. Variabel kinematik semen beku juga menunjukkan perbedaan antar pengencer. Nilai kecepatan tertinggi ditunjukkan oleh AndroMed® (VCL 161,8 ± 3,70 µm/s; VSL 74,42 ± 2,52 µm/s; VAP 84,02 ± 2,54 µm/s). Nilai ALH tertinggi diperoleh pada pengencer TKT (3,21 ± 0,11 µm), menunjukkan amplitudo gerak kepala yang besar namun arah gerak kurang stabil. Nilai BCF tertinggi diperoleh pada BoviFree® dan AndroMed® (sekitar 15-16 Hz) yang menandakan efisiensi pergerakan flagelum. Steridyl® menunjukkan nilai LIN (0,492 ± 0,01) dan STR (0,863 ± 0,00) tertinggi, menggambarkan arah gerak spermatozoa yang lebih lurus dan terkontrol. Hasil uji longivitas menunjukkan penurunan motilitas seiring waktu inkubasi, namun pengencer komersial mampu mempertahankan motilitas lebih lama dibandingkan dengan TKT yang ditandai perbedaan signifikan pada jam ke 4. Motilitas total masih bertahan pada 18,47 ± 2,61% (AndroMed®), 16,08 ± 3,32% (BoviFree®), 14,57 ± 4,44% (Steridyl®), sedangkan TKT hanya 7,77 ± 2,37% pada empat jam inkubasi pasca-thawing. Pola yang sama terlihat pada motilitas progresif. Nilai recovery rate motilitas progresif menunjukkan perbedaan nyata antar pengencer (P = 0,007), dengan Steridyl® memiliki nilai tertinggi (64,11%), menunjukkan kemampuan terbaik dalam mempertahankan motilitas pasca-pembekuan dan thawing. Hasil penelitian ini menunjukkan bahwa komposisi pengencer berperan penting terhadap freezability spermatozoa. Steridyl® mengandung lipoprotein kuning telur steril, memberikan perlindungan paling efektif melalui stabilisasi lipid membran, penurunan efek cold shock, dan pemeliharaan fungsi mitokondria. Pengencer berbasis lesitin kedelai seperti AndroMed® dan dan lesitin sintetis seperti BoviFree® mampu mempertahankan kecepatan gerak (VCL, VSL, VAP) dan BCF yang tinggi, namun kurang optimal dalam menjaga viabilitas dan motilitas progresif. Pengencer homemade TKT kurang dapat melindungi spermatozoa saat kriopreservasi. Penelitian ini menyimpulkan karakteristik dan kinematik spermatozoa semen segar domba lokal menunjukkan variasi antar individu. Kualitas spermatozoa semen beku terbukti dipengaruhi oleh jenis pengencer. Steridyl® paling optimal dalam mempertahankan kualitas spermatozoa selama proses pembekuan, sedangkan AndroMed® paling optimal dalam variabel kinematik spermatozoa. Longivitas dan recovery rate spermatozoa terbukti berbeda antar pengencer, dengan pengencer komersial menunjukkan longivitas yang lebih baik dibandingkan pengencer homemade, dan Steridyl® memberikan recovery rate tertinggi dibandingkan pengencer lain yang diujikan.

The decline in the local Indonesian sheep population necessitates the implementation of artificial reproductive technologies to sustain productivity and enhance the success of insemination. The success of artificial insemination (AI) largely depends on semen quality, particularly the ability of spermatozoa to survive the freezing process (freezability). This quality is strongly influenced by the type of diluent used to protect the sperm membranes from cryogenic stress. This study was conducted to analyze the effects of homemade and commercial diluents on the quality, kinematics, longevity, and recovery rate (RR) of frozen spermatozoa from local sheep, using an objective analytical approach through computer assisted sperm analysis (CASA). The study involved three local rams, each subjected to six repetitions. Evaluations were performed on both fresh and frozen semen using four different diluents: Tris Egg Yolk (TEY, homemade), AndroMed®, BoviFree®, and Steridyl®. The observed variables included macroscopic characteristics, motility, viability, acrosome integrity, abnormalities, sperm concentration, and kinematic variables such as curvilinear velocity (VCL), straight line velocity (VSL), average pathway velocity (VAP), amplitude of lateral head displacement (ALH), beat cross frequency (BCF), linearity (LIN), and straightness (STR). Fresh semen exhibited normal macroscopic characteristics with an average volume of 1.32 ± 0.13 mL, creamy color, thick consistency, and pH of 6.64 ± 0.05. The total and progressive motility were 68.11 ± 3.83% and 62.09 ± 4.07%, respectively, with viability of 53.61 ± 3.57%, acrosome integrity of 95.22 ± 1.14%, and abnormalities of 23.04 ± 2.64%. The average sperm concentration was 2,754.65 ± 178.94 ×106/mL (CASA method). Kinematic assessment of fresh semen indicated VCL of 198.82 ± 8.47 µm/s, VSL of 88.80 ± 5.84 µm/s, VAP of 103.25 ± 6.12 µm/s, ALH of 3.88 ± 0.11 µm, BCF of 12.73 ± 0.45 Hz, LIN of 0.46 ± 0.01, and STR of 0.83 ± 0.01. These values indicate highly active spermatozoa with stable movements and strong fertilisation potential. Spermatozoa after freezing experienced a decline in quality across all diluents. Steridyl® produced the highest total motility (44.98 ± 4.72%), progressive motility (41.07 ± 4.56%), and viability (42.56 ± 4.35%), exceeding the minimum standard set by the Indonesian National Standard (=40%). AndroMed® and BoviFree® demonstrated moderate performance, whereas TEY showed the lowest results. The kinematic variables of frozen semen also differed among diluents. The highest velocity values were observed in AndroMed® (VCL 161.8 ± 3.70 µm/s; VSL 74.42 ± 2.52 µm/s; VAP 84.02 ± 2.54 µm/s). The highest ALH value was recorded in TEY (3.21 ± 0.11 µm), indicating a wider head movement amplitude but lower directional stability. The highest BCF values were found in BoviFree® and AndroMed® (approximately 15-16 Hz), reflecting efficient flagellar activity. Steridyl® exhibited the highest LIN (0.492 ± 0.01) and STR (0.863 ± 0.00), suggesting more linear and controlled sperm movement. The longevity test demonstrated a time dependent decrease in motility however, commercial diluents maintained sperm motility for longer periods compared to TEY, as indicated by significant differences at the 4-hour incubation period. After four hours post-thawing, total motility remained at 18.47 ± 2.61% (AndroMed®), 16.08 ± 3.32% (BoviFree®), and 14.57 ± 4.44% (Steridyl®), whereas TEY showed only 7.77 ± 2.37%. Progressive motility followed a similar trend. The recovery rate (RR) of progressive motility differed significantly among diluents (P = 0.007), with Steridyl® showing the highest RR (64.11%), indicating its superior ability to preserve motility after freezing and thawing. The findings confirm that the composition of the diluent plays a crucial role in determining sperm freezability. Steridyl®, which contains sterilized egg yolk lipoproteins, provided the most effective protection through membrane lipid stabilization, reduction of cold shock effects, and preservation of mitochondrial function. Soy lecithin based diluents such as AndroMed® and synthetic lecithin based BoviFree® were able to maintain high movement speed (VCL, VSL, VAP) and BCF but were less effective in sustaining viability and progressive motility. The homemade TEY diluent was suitable for handling fresh semen but insufficiently protective under cryogenic conditions. The study concludes that the characteristics and kinematics of fresh ram spermatozoa exhibit inter-individual variation. The quality of frozen thawed spermatozoa was demonstrably influenced by the type of diluent used. Steridyl® was the most effective in preserving sperm quality during cryopreservation, whereas AndroMed® yielded the most optimal results for sperm kinematic variables. Sperm longevity and recovery rate differed among diluents, with commercial diluents demonstrating superior longevity compared to the homemade diluent, and Steridyl® providing the highest recovery rate among all treatments.