三倍化是一種廣泛使用的生產(chǎn)不育魚(yú)種群的方法。生產(chǎn)三倍體魚(yú)最有效的方法是施加壓力沖擊（Pandian 和 Koteeswaran引文1998).然而，三倍體紅羅非魚(yú)是由熱沖擊和冷沖擊產(chǎn)生的（Pradeep、Srijaya、Bahuleyan 等人。引文2012;普拉迪普、斯里賈亞、帕皮尼等人。引文2012).為不同魚(yú)類的實(shí)驗(yàn)處理選擇合適的技術(shù)參數(shù)并不容易，需要大量的經(jīng)驗(yàn)和技能來(lái)檢查最終結(jié)果，例如使用細(xì)胞遺傳學(xué)或細(xì)胞術(shù)方法對(duì)魚(yú)類實(shí)驗(yàn)組進(jìn)行倍性驗(yàn)證（Benfey 等人。引文1984;約翰斯通和林肯引文1986).在紅羅非魚(yú)中，根據(jù)紅細(xì)胞的核體積、細(xì)胞質(zhì)體積和細(xì)胞核表面積進(jìn)行三倍體個(gè)體的鑒定（Pradeep 等人。引文2011).最合適的溪鱒魚(yú)倍性驗(yàn)證方法，Salvelinus fontinalis，似乎是通過(guò)血涂片進(jìn)行紅細(xì)胞尺寸研究（Woznicki 和 Kuzminski引文2002).

在由壓力或熱沖擊產(chǎn)生的三倍體魚(yú)中，觀察到紅細(xì)胞、血紅蛋白和紅細(xì)胞指數(shù)的改變（Benfey 和 Sutterlin引文1984年b;本菲等人。引文1984;本菲引文1999;Strunjak-Perovic 等人。引文2003;Wlasow 等人。引文2004;多拉夫山等人。引文2008;王等人。引文2010;Wlasow 和 Fopp-Bayat引文2011).三倍化還影響白細(xì)胞、免疫防御反應(yīng)和應(yīng)激反應(yīng)的變化（Svobodová 等人。引文1998,引文2001本菲和比隆引文2000;貝亞等人。引文2005;馬克西姆引文2008;弗雷澤等人。引文2012).

在三倍體溪鱒魚(yú)的外周血中，與對(duì)照組相比，細(xì)胞核分裂的紅細(xì)胞百分比在統(tǒng)計(jì)學(xué)上顯著更高（19.1% 對(duì) 0.3%）（Wlasow 等人。引文2004).然而，在這項(xiàng)研究中，顯示了受精后 20 分鐘施加的沖擊引起的三倍化的影響，但沒(méi)有檢查其他技術(shù)參數(shù)的影響。因此，本研究的首要目的是研究從受精到壓力休克的時(shí)間差異對(duì)三倍體溪鱒魚(yú)血細(xì)胞改變的影響。該研究的第二個(gè)目的是估計(jì)測(cè)定溪鱒魚(yú)三倍體外周血中細(xì)胞核分裂的紅細(xì)胞百分比所需的最小紅細(xì)胞數(shù)量。

材料和方法

Material and methods

Fish used for this study were cultured at the Salmonid Research Department in Rutki (Institute of Inland Fisheries in Olsztyn, Poland). Both diploids (2n) and triploids (3n) came from the same lot of eggs. Triploid brook trout Salvelinus fontinalis (Mitchill) were obtained by using hydrostatic pressure shock of 9500 psi (65.5 × 103 kPa) on eggs at 10°C (Deeley and Benfey Citation1995). The pressure shock duration was 5 minutes, but time from fertilisation to shock varied in the experimental groups, and ranged from 22.5 min (group 3N-1) to 62.5 min. (group 3N-8, Table 1).The diploid (control – 2N, 3N) received no pressure shock. Fish of the experimental groups were kept in separate tanks supplied with river water. During the experiment prophylactic chloramine baths were used twice a week. These baths are carried out during each rearing of fish at the Salmonid Research Department in Rutki (Institute of Inland Fisheries in Olsztyn, Poland) due to the presence of pathogens in the water supply hatchery and rearing system. Fish were fed granulate feed BioMar (Denmark). Survival from hatching to the collection of blood is shown in Table 1. Two months after hatching blood was collected from the caudal vessels to heparinised syringes. Propiscin (0.2% etomidate, Inland Fisheries Institute in Olsztyn, Poland) was used as an anaesthetic (0.5 ml l^?1 of water for 10 min) in all experimental groups. Fish expected to be triploids (eight experimental groups, Table 1) and diploids (20 specimens) were used for preparing blood smears. Smears were fixed in 95% methanol for 3 minutes, left to air dry and stained with 20% Giemsa solution for 15 minutes. The percentage of erythrocytes with divided nuclei was determined based on the observation of 300 cells from two slides for fish. For the study of white blood cells 200 cells were analysed, and the following forms have been studied: normal lymphocytes, Rieder’s lymphocytes (cells with divided nuclei), and neutrophil granulocytes (NGs) with 2, 3, 4 and ≥?5 segments of nuclei. The identification was based on the leukocyte descriptions published by Lehmann and Stürenberg (Citation1975).

用于本研究的魚(yú)是在魯特基的鮭魚(yú)研究部（波蘭奧爾什丁內(nèi)陸漁業(yè)研究所）養(yǎng)殖的。二倍體 （2n） 和三倍體 （3n） 都來(lái)自同一批卵。三倍體溪鱒魚(yú) Salvelinus fontinalis （Mitchill） 是通過(guò)在 10°C 下對(duì)雞蛋進(jìn)行 9500 psi （65.5 × 103 kPa） 的（上海瑾瑜出售）靜水壓力機(jī)沖擊獲得的 （Deeley 和 Benfey）引文1995).壓力休克持續(xù)時(shí)間為5 min，但實(shí)驗(yàn)組從受精到休克的時(shí)間各不相同，從22.5 min（3N-1組）到62.5 min不等（3N-8組，表1).二倍體（對(duì)照 – 2N、3N）沒(méi)有受到壓力沖擊。實(shí)驗(yàn)組的魚(yú)被飼養(yǎng)在單獨(dú)的水箱中，供水。在實(shí)驗(yàn)期間，每周使用兩次預(yù)防性氯胺浴。由于供水孵化場(chǎng)和飼養(yǎng)系統(tǒng)中存在病原體，這些浴是在 Rutki 的鮭魚(yú)研究部（波蘭奧爾什丁內(nèi)陸漁業(yè)研究所）每次養(yǎng)魚(yú)期間進(jìn)行的。魚(yú)被喂食顆粒飼料 BioMar（丹麥）。從孵化到采血的存活率見(jiàn)表1.孵化后兩個(gè)月，從尾部血管收集血液到肝素注射器中。使用普羅匹辛（0.2% 依托咪酯，波蘭奧爾什丁內(nèi)陸漁業(yè)研究所）作為麻醉劑（0.5 毫升升^?1水 10 分鐘）在所有實(shí)驗(yàn)組中。預(yù)計(jì)為三倍體的魚(yú)（八個(gè)實(shí)驗(yàn)組，表1）和二倍體（20個(gè)標(biāo)本）用于制備血涂片。將涂片固定在95%甲醇中3分鐘，風(fēng)干并用20%吉姆薩溶液染色15分鐘。根據(jù)對(duì)兩張魚(yú)類載玻片的 300 個(gè)細(xì)胞的觀察，確定具有分裂細(xì)胞核的紅細(xì)胞百分比。為了研究白細(xì)胞，分析了 200 個(gè)細(xì)胞，并研究了以下形式：正常淋巴細(xì)胞、里德淋巴細(xì)胞（細(xì)胞核分裂的細(xì)胞）和具有 2、3、4 和 ≥ 5 段細(xì)胞核的中性粒細(xì)胞 （NG）。鑒定基于 Lehmann 和 Stürenberg 發(fā)表的白細(xì)胞描述（引文1975).

Simultaneously chromosome preparations (10 specimens from each experimental group) and the erythrocyte nuclei major axis measurements (all fish from each treatment) were done using procedure described by Woznicki and Kuzminski (Citation2002) in order to confirm fish ploidy. Differential cell counts were made under a 1000 × magnification.

Statistical analyses were performed on data from microscopic observations as the actual number of segmented nuclei in analysed preparations. In Tables 2 and 3 data were summarised in the form of percentages. All statistical analyses were evaluated at a significance level of 0.05. The chi-square test was used to evaluate the differences in proportion of “abnormal” to normal erythrocytes and leukocytes in triploid and diploid trout. Statistical differences between the observed number of red blood cells with segmented nucleus in the groups of 2N and 3N fish were calculated based on an alternative non-parametric analysis of variance – ANOVA Kruskal–Wallis (α = 0.05). In order to investigate how many nuclei should be counted to show the dependence of the number of nuclei divided by ploidy the Friedman ANOVA test was used. Data were collected from 71 fish, step by step as follows: 50 nuclei were observed and selected as normal and divided, and then the next 50 nuclei were observed and also selected as normal and divided (two or more parts), after that the cyclic analysis of next 50 nuclei was conducted. A total of 300 nuclei were counted. It was hypothesised that the results obtained for the count of 50, 100, 150, 200, 250 and 300 nuclei did not differ significantly and therefore the study of ploidy is sufficient to perform only counts of 50 nuclei.

同時(shí)使用 Woznicki 和 Kuzminski 描述的程序進(jìn)行染色體制備（每個(gè)實(shí)驗(yàn)組 10 個(gè)標(biāo)本）和紅細(xì)胞核長(zhǎng)軸測(cè)量（來(lái)自每個(gè)處理的所有魚(yú)）（引文2002）以確認(rèn)魚(yú)的倍性。在 1000 ×放大倍率下進(jìn)行差異細(xì)胞計(jì)數(shù)。

對(duì)來(lái)自顯微觀察的數(shù)據(jù)進(jìn)行統(tǒng)計(jì)分析，作為分析制劑中分段細(xì)胞核的實(shí)際數(shù)量。在表格中2和3數(shù)據(jù)以百分比的形式匯總。所有統(tǒng)計(jì)分析均以 0.05 的顯著性水平進(jìn)行評(píng)估。采用卡方檢驗(yàn)評(píng)估三倍體和二倍體鱒魚(yú)中“異常”與正常紅細(xì)胞和白細(xì)胞比例的差異。根據(jù)替代非參數(shù)方差分析 - 方差分析 Kruskal-Wallis （α = 0.05） 計(jì)算 2N 和 3N 魚(yú)組中觀察到的具有分段細(xì)胞核的紅細(xì)胞數(shù)量之間的統(tǒng)計(jì)差異。為了研究應(yīng)該計(jì)算多少個(gè)細(xì)胞核以顯示細(xì)胞核數(shù)除以倍性的依賴性，使用了弗里德曼方差分析檢驗(yàn)。從71條魚(yú)中收集數(shù)據(jù)，逐步如下：觀察50個(gè)細(xì)胞核，選擇正常并分裂，然后觀察接下來(lái)的50個(gè)細(xì)胞核，也選擇為正常并分裂（兩個(gè)或多個(gè)部分），然后對(duì)接下來(lái)的50個(gè)細(xì)胞核進(jìn)行循環(huán)分析。總共計(jì)算了 300 個(gè)細(xì)胞核。據(jù)推測(cè)，對(duì) 50、100、150、200、250 和 300 個(gè)細(xì)胞核的計(jì)數(shù)獲得的結(jié)果沒(méi)有顯著差異，因此倍性研究足以僅對(duì) 50 個(gè)細(xì)胞核進(jìn)行計(jì)數(shù)。