Functional and Fertility Outcomes of Two Milk‐Based Extenders for Short‐Term Storage of Ram Semen at 15°C

ABSTRACT

The efficiency of AI in sheep depends partly on maintaining sperm function during storage. This study compared INRA 96 and a skim milk‐based formulation for storing ram semen at 15°C for 6 h, evaluating sperm quality and IVF and field fertility outcomes. INRA 96 showed higher VCL, ALH, and sperm viability after 6 h of storage. However, cleavage rate, blastocyst rate, and blastocyst cell number after IVF, as well as field fertility and prolificacy, did not differ between extenders. These results indicate that the skim milk‐based formulation preserved reproductive outcomes at levels comparable to INRA 96, supporting its suitability as a cost‐effective alternative for routine AI programs.

1. Introduction

Artificial insemination (AI) in sheep usually involves vaginal deposition of liquid‐stored semen at 15°C for up to 6–8 h (Alvarez et al. 2019), although fertility remains variable and often suboptimal (Palacín et al. 2012). Extender composition is an important factor affecting sperm function during storage and, therefore, AI success (Khalifa et al. 2013; O'Hara et al. 2010; Paulenz et al. 2003).

INRA 96 is a commercially available extender widely used for short‐term storage of ram semen in Mediterranean countries (Neila‐Montero et al. 2024). Skim milk‐based extenders have also been traditionally employed in small ruminants due to their protective properties and low cost (López‐Sáez et al. 2000). However, direct comparisons under standardized conditions integrating sperm function and fertilization outcomes remain limited. Accordingly, this study compared INRA 96 and a skim milk‐based formulation for storing ram semen at 15°C for 6 h, evaluating sperm quality, in vitro fertilization, and field fertility outcomes.

2. Materials and Methods

2.1. Semen Collection and Processing for Sperm Evaluation and IVF

Individual ejaculates from three Manchega rams housed at the ETSIAMB experimental farm (ES008007; Albacete, Spain) were collected by artificial vagina and pooled after confirming minimum quality standards: ≥ 0.5 mL, ≥ 3000 × 10⁶ sperm/mL, and ≥ 4 mass motility. For evaluation of sperm quality and IVF, the pooled sample was divided and diluted either in INRA 96 (COM) or skim milk‐based extender (SM; 100 mg/mL skim milk powder in Milli‐Q water with 0.03 mg/mL penicillin and 0.04 mg/mL gentamicin) to 1600 × 10⁶ sperm/mL. Samples were cooled from 30°C to 15°C in a temperature‐controlled water bath (−0.5°C/min) and maintained under these conditions for 6 h. Sperm quality was evaluated at 0, 3, and 6 h of storage, and in vitro fertilization (IVF) was performed at 6 h. The entire experimental procedure was repeated three times.

2.2. Sperm Quality Assessment

2.2.1. Sperm Motility

Sperm motility was assessed by SCA v6.5.0.44 after dilution to 20 × 10⁶ sperm/mL with the same extender and pre‐warming at 37°C for 5 min, using a Makler chamber under phase‐contrast microscopy (10× objective) with a heated stage. At least 400 sperm per sample were analysed, recording curvilinear velocity (VCL, μm/s), average path velocity (VAP, μm/s), linearity (LIN, %), and amplitude of lateral head displacement (ALH, μm). Total and progressive motility (TM and PM, %) were defined as sperm with VCL > 15 and 45 μm/s, respectively.

2.2.2. Sperm Viability

Sperm viability was assessed by eosin‐nigrosin staining, prepared as described by Soler et al. (2025) Smears were examined under bright‐field microscopy (40× objective), classifying 100 sperm as viable or non‐viable based on eosin exclusion.

2.3. IVF, Embryo Culture, and Blastocyst Quality Assessment

Cumulus‐oocyte complexes were recovered from post‐mortem ovaries from sheep, matured, fertilized, and cultured as previously described by Martín‐Maestro et al. (2025). Cleavage and blastocyst rates were assessed at 48 h and day 8 post‐insemination. Day‐8 blastocysts were fixed, stained with DAPI, and examined under epifluorescence microscopy to count total cell number.

2.4. AI Assay

A total of 3790 Manchega ewes from 41 farms were randomly assigned to cervical AI with 359 ejaculates from 72 rams obtained by artificial vagina and extended in COM or SM at 1600 × 10⁶ sperm/mL. Estrus was synchronized using a standard progestagen‐eCG protocol, and AI were performed 54 ± 2 h later using 400 × 10⁶ sperm doses stored at 15°C for ≤ 6 h. Fertility was determined from lambings 137–154 days after AI, and prolificacy as lambs born per AI‐derived lambing. All procedures for sperm collection and processing for AI, together with AI procedures, were carried out by technicians from the Centro Nacional de Selección y Reproducción Animal (CERSYRA) and the Asociación Nacional de Criadores de Ganado Ovino Selecto de Raza Manchega (AGRAMA), as is routinely done.

2.5. Statistical Analysis

Data were analysed using IBM SPSS Statistics v29.0.2.0. Homogeneity of variance was verified using Levene's test. Sperm quality parameters were analysed by GLM, including extender, storage time, and their interaction, followed by Bonferroni post hoc tests. IVF outcomes were compared using an independent samples t‐test. Field fertility and prolificacy were analysed using mixed models (GLMM and LMM, respectively), with extender as a fixed effect and farm and ram as random effects. Results are expressed as mean ± SEM, and asterisks indicate significant changes (*p < 0.05; **p < 0.01; ***p < 0.001).

3. Results

3.1. Sperm Quality Assessment

TM was higher in SM at 0 h. COM showed higher VCL (3 and 6 h) and ALH (all time points), whereas SM showed higher LIN at 0 and 6 h. No differences were observed for PM and VAP (Figure 1).

FIGURE 1.

Sperm motility of ram semen stored at 15°C for 6 h in two extenders: INRA 96 (COM) and skim milk (SM). Data are presented as mean ± SEM (n = 3 independent biological replicates, each consisting of pooled semen from three rams). Asterisks indicate significant differences between extenders (*p < 0.05; **p < 0.01; ***p < 0.001).

Sperm viability after 6 h was higher in COM than in SM (Figure 2).

FIGURE 2.

Sperm viability (%) of ram semen stored at 15°C for 6 h in two extenders: INRA 96 (COM) and skim milk (SM). Data are presented as mean ± SEM (n = 3 independent biological replicates, each consisting of pooled semen from three rams). Asterisk indicates significant difference between extenders (p < 0.05).

3.2. IVF and AI Assays

Cleavage and blastocyst rates after IVF did not differ between extenders, with values of 77.71% ± 8.82% and 34.14% ± 2.31% for COM, and 75.08% ± 5.83% and 28.18% ± 2.73% for SM, respectively. Blastocyst quality, assessed as total cell number, was also similar between treatments (95.00 ± 6.15 for COM vs. 104.24 ± 10.52 for SM) (Figure 3).

FIGURE 3.

Representative confocal images of DAPI‐stained blastocyst obtained after IVF using ram semen stored at 15°C for 6 h in two extenders: INRA 96 (COM) and skim milk (SM).

Likewise, no differences were observed in field fertility (27.01% ± 2.00% vs. 29.00% ± 2.10%) or prolificacy (1.46 ± 0.03 vs. 1.47 ± 0.03 lambs/ewe).

4. Discussion

This study compared INRA 96 and a skim milk‐based formulation for storing ram semen at 15°C for 6 h, integrating functional sperm assessment, in vitro embryo development and quality, and field fertility outcomes.

INRA 96 showed higher sperm motility (VCL and ALH) and viability after 6 h, suggesting enhanced membrane preservation. Similar findings have been reported in stallions, where INRA 96, based on purified native phosphocaseinate (NPPC), improved sperm motility and survival during storage at 15°C compared with conventional milk‐based extenders (Batellier et al. 1997), and progressive motility during storage at 4°C compared with the skim milk‐based extender Botusemen (de Guevara Ojeda et al. 2025). Casein micelles are the main protective components of milk during semen storage (Manjunath 2012), interacting with seminal plasma BSP proteins and modulating lipid efflux from the sperm membrane (Lusignan et al. 2011). Since both extenders contain caseins, the observed differences may be related to the standardized NPPC fraction (α‐, β‐, and κ‐caseins in defined proportions) in INRA 96 rather than to distinct protective mechanisms.

However, these functional differences were not reflected in embryo development and blastocyst quality after IVF or fertility and prolificacy after AI. Similar discrepancies between sperm quality parameters and fertility outcomes have been reported previously (Arando et al. 2019; Paulenz et al. 2003), highlighting the limited predictive value of conventional semen assessments (Rodríguez‐Martínez 2006). While marked differences in ram sperm quality parameters affect field fertility (Vicente‐Fiel et al. 2014), subtler variations between treatments may not translate into altered reproductive performance.

In conclusion, when Manchega ram semen is stored at 15°C for 6 h, the skim milk‐based formulation represents a cost‐effective alternative to INRA 96 without compromising reproductive outcomes. Further studies should evaluate its performance during longer storage periods and in additional sheep breeds.

Author Contributions

Conceptualization: M.N.‐M., A.J.S.; Methodology and Investigation: M.N.‐M., M.I.‐C., J.H.‐S., E.D.‐H., C.V.‐G., J.A.L.‐G., M.S., R.G., Ó.G.‐G.; Formal analysis and Data curation: M.N.‐M.; Writing – original draft: M.N.‐M.; Writing – review and editing: M.I.‐C., A.J.S.; Supervision and Project administration: A.J.S.; Funding acquisition: J.J.G. and A.J.S. All authors approved the final manuscript.

Funding

This work was financed by the GC4Sheep project (EAFRD—MAPA—PNDR; NextGenerationEU). E.D.‐H. was supported by NextGenerationEU (2025‐INVGO‐13367) and C.V.‐G. by MINECO (PRE2022‐102432).

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.