Coconut Water as an Extender Component for Cooled Equine Sperm

Highlights

• Plasma membrane integrity of cooled equine sperm was maintained with ACP-105.

• Coconut water–based extenders do not provide satisfactory results in preserving the parameters of equine cooled semen.

• Addition of milk or egg yolk with ACP-105 did not improve sperm parameters of cooled equine semen.

Abstract

The aim of this study was to evaluate the effect of coconut water as a component of extender in different formulations for cooling equine sperm. One ejaculate of fourteen stallions was collected. Sperm was diluted to 50 × 10⁶ sperm/mL using five different extenders: ACP-105: powdered coconut water extender (ACP-105, ACP Biotecnologia, Brazil); ACP-Milk: ACP-105 + 20 g/L of skimmed milk; ACP-EY 2.5%: ACP-105 + 2.5% of egg yolk; ACP-EY 5%: ACP-105 + 5% of egg yolk; and BotuSêmen (Botupharma, Botucatu, Brazil) and cooled in passive cooling device (BotuFlex, Botupharma, Botucatu, Brazil) at 5 and 15°C for 24 hours. Sperm kinetics and plasma membrane integrity (PMI) were evaluated by computer-assisted sperm analysis and fluorescence staining, respectively, at T0 (before cooling) and T24 (24 hours after cooling). Sperm kinetics did not differ at T0 among groups (P > .05); however, at T24, these parameters were significantly lower in ACP-105 (5°C, total motility [TM]: 9.2 ± 3.6%; progressive motility [PM]: 2.7 ± 1.6%; percentage of fast-moving spermatozoa [RAP]: 4.8 ± 3.0%; 15°C, TM: 10.6 ± 3.0%; PM: 1.1 ± 0.5%; RAP: 4.8 ± 1.9%) and ACP-EY 5% (5°C, TM: 28.0 ± 6.3%; PM: 5.7 ± 1.8%; RAP: 15.9 ± 6.0%; 15°C, TM: 30.0 ± 6.0%; PM: 6.9 ± 2.1%; RAP: 17.6 ± 5.3%) compared with BotuSêmen (5°C, TM: 66.2 ± 5.6%; PM: 21.1 ± 2.8%; RAP: 53.9 ± 6.1%; 15°C, TM: 63.4 ± 5.4%; PM: 17.2 ± 2.8%; RAP: 51.4 ± 6.3%) (P < .05). All groups exhibited similar PMI at tested moments and cooling temperatures (5°C: 83%; 15°C: 84%) (P > .05). Further studies are necessary to evaluate powdered coconut water in different compositions of sperm extender; however, coconut-based extender as used in this study was not an alternative to preserve sperm parameters of cooled equine sperm.

Introduction

In tropical regions, coconut water is a natural and abundant resource. This comprises salts, proteins, sugars, vitamins, neutral fats, cell division inducers, and various electrolytes [1], which provides nutrients necessary for cell preservation [2], [3]. Studies have shown that a coconut water–based extender may be an alternative for sperm preservation [4], [5]; however, there are some disadvantages, such as the inability to store coconut water for long period and its limited availability in some regions of the world. Furthermore, the biochemical constitution of each coconut differs, and this can directly affect the ability of the extender to preserve spermatozoa.

Some studies were conducted to develop a powdered coconut water–based extender (ACP-105; ACP Biotecnologia, Fortaleza, Ceará, Brazil), which has been reported to be an alternative for sperm preservation in goats [5], [6], boars [7], dogs [8], [9], and stallions [10]. Although the powdered coconut water–based extender has been successfully used for sperm preservation of several species, experiment with stallion sperm is limited.

Cooled equine sperm for artificial insemination has become a widespread biotechnology in the horse industry for decades [11]. It plays an important role in this business because it enables trade and shipment of genetic material over long distances with maintenance of satisfactory fertility rates [12]. Cooling process reduces sperm metabolism and consequently increases cell viability [11], [13]. However, during the course of cooling, spermatozoa are exposed to fast structural changes, which can promote damages to the sperm cell, compromising cell function by reduction of plasma membrane fluidity and alteration of lipid and protein organization [14], [15], [16]. Therefore, the aim of this study was to evaluate the efficiency of the powdered coconut water–based extender in preserving cooled equine sperm.